HA-1 epitopes and uses thereof

ABSTRACT

Peptide sequences constituting T-cell epitopes of minor Histocompatibility antigen, HA-1. HA-1 is associated with Graft versus Host Disease. The peptides and their derivatives find many uses, for instance, in bone marrow transplantation, organ transplantation and in treatment of leukemia and non-hematopoietic tumors. The peptide and/or its derivatives can be incorporated in vaccines, in pharmaceutical formulations and they can be used in diagnostic test kits. HA-1 is expressed by non-hematopoietic tumor cells. While absent in normal epithelial cells, tumor cells and tumor cell lines, particularly from epithelial origin, express HA-1 and are recognized by HA-1 cytotoxic T-cells. The invention provides means and methods for HA-1 specific immunotherapy for HA-1-positive patients with non-hematopoietic tumor cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 10/623,176, filed Jul. 18, 2003, which application is acontinuation in part of co-pending application U.S. Ser. No. 09/489,760,filed on Jan. 21, 2000, the contents of the entirety of each of whichare incorporated herein by this reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5) -SEQUENCE LISTING SUBMITTEDON COMPACT DISC

Pursuant to 37 C.F.R. §1.52(e)(1)(iii), a compact disc containing anelectronic version of the Sequence Listing has been submittedconcomitant with this application, the contents of which are herebyincorporated by reference. A second compact disc is submitted and is anidentical copy of the first compact disc. The discs are labeled “copy 1”and “copy 2,” respectively, and each disc contains one file entitled“2183-6047 seq list.txt” which is 35 KB and created on Jan. 9, 2006.

TECHNICAL FIELD

The invention relates generally to biotechnology, and more particularlyto the field of cellular immunology.

BACKGROUND

Bone marrow transplantation (BMT) finds its application in the treatmentof, for instance, severe aplastic anemia, leukemia and immune deficiencydiseases.

In the early days of this technique, many transplants failed throughrejection of the graft by the host. Transplants that did succeed,however, often led to an immune response by lymphocytes present in thegraft against various tissues of the host (GvHD). It is now known thatthe GvHD response is mainly due to the presence of majorhistocompatibility (H) antigens which present a transplantation barrier.Therefore, it is now routine practice to graft only HLA-matchedmaterials (either from siblings or unrelated individuals) resulting in amuch improved rate of success in bone marrow transplantation. However,despite this improvement, as well as improvements in pre-transplantationchemotherapy or radiotherapy and the availability of potentimmuno-suppressive drugs, about 20 to 70% of the treated patients stillsuffer from GvHD (the percentage is age and bone marrow donordependent). To avoid GvHD, it has been suggested to remove the cells(mature T-cells) causing the reaction from the graft. This removal,however, often leads to graft failure or to recurrence of the originaldisease. The cells responsible for GvHD are also the cells which oftenreact against the original aberrant cells in, for instance, leukemia(Graft versus Leukemia response).

Since BMT is nowadays mainly carried out with HLA matched grafts, theGvHD which still occurs must be caused by another group of antigens. Itis very likely that the group of so called minor H antigens (mHag),which are non-MHC encoded histocompatibility antigens (unlike the majorH antigens), are at least partially responsible for the remainingincidence of GvHD. mHags were originally discovered in congeneic strainsof mice in tumor rejection and skin rejection studies. In mice, the useof inbred strains has shown that mHag are encoded by almost 50 differentallelicly polymorphic loci scattered throughout the genome. In humans,although cumbersome to identify, mHag have been shown to exist, buttheir overall number and complexity remains uncertain. Minor H antigensare most likely quite different from each other and quite different frommajor H antigens; mHags are probably a diverse and elusive group offragments of molecules which are participating in various cellularhousekeeping functions. Their antigenicity may come very incidentally,as naturally processed fragments of polymorphic proteins that associatewith MHC products. Some of the mHag appear to be widely expressed onvarious tissues throughout the body whereas others show limited tissuedistribution.

One of the better known minor histocompatibility antigens is the H-Yantigen. H-Y is an mHag that can lead to rejection of HLA-matched maleorgan and bone marrow grafts by female recipients, and to a higherincidence of GvHD in female-to-male grafts, particularly if the femaledonor had been previously pregnant. The H-Y antigen may also play a rolein spermatogenesis. The human H-Y antigen is an eleven-residue peptidederived from SMCY, an evolutionary conserved Y chromosomal protein.

Another well-known mHag that can lead to GvHD is the HA-2 antigen. Thehuman HA-2 antigen is a nine-residue peptide likely derived from aclass-I myosin. However, the nature of the HA-1 antigen, responsible fora majority of current cases of GvHD has remained elusive so far. Humanbone marrow transplants performed as therapeutic treatment of severeaplastic anemia, leukemia and immune deficiency disease became availablein the seventies.

For the present, the long-term results of allogeneic bone marrowtransplantation (BMT) have greatly improved due to the use ofHLA-matched siblings as marrow donors, advanced pre-transplantchemo/radiotherapy, the use of potent immunosuppressive drugs as GvHDprophylaxis, better antibiotics and isolation procedures. Nonetheless,the results of clinical BMT reveal that the selection of MHC identicaldonors/recipients is not a guarantee of avoidance of GvHD ordisease-free survival even when donor and recipient are closely related.Allogeneic BMT, especially in adults, results, depending on the amountof T-cell depletion of the graft, in up to 80% of the cases in GvHD. Inthe HLA genotypically identical situation it amounts to 15 to 35%,whereas in the phenotypical HLA matched recipient/donor combinations,the occurrence of GvHD is significantly higher, i.e., 50 to 80%.Disparities for minor Histocompatibility antigens (mHag) between donorand recipient constitute a potential risk for GvHD or graft failure,which necessitate life long pharmacologic immunosuppression of organ andbone marrow transplant recipients.

It is also believed that mHag are involved in the “beneficial” sideeffect of GvHD, i.e., the Graft-versus-Leukemia activity. Severalreports demonstrated the presence of anti-host mHag-specific cytotoxicT-lymphocytes (“CTLs”) in patients suffering from GvHD after HLAgenotypically identical BMT. In our laboratory, much effort was put intothe further characterization of a number of anti-host mHag-specificCTLs. Hereto, CTL clones specific for host mHag were isolated from theperipheral blood (PBL) of patients suffering from severe GvHD. mHagHA-1-specific CD8⁺ CTL clones were originally obtained afterrestimulation of in vivo primed PBLs from three patients suffering fromGvHD after HLA identical but mHag nonidentical BMT. The post-BMT CTLlines were cloned by limiting dilution, resulting in the isolation of alarge number of mHag-specific CTL clones. Subsequent immunogeneticanalyses revealed that the CTL clones (as described above) identifiedfive non-sex-linked mHag, designated HA-1, -2, -3, -4, -5, which arerecognized in a classical MHC restricted fashion. mHag HA-3 wasrecognized in the presence of HLA-A1 and mHag HA-1, -2, -4 and -5 werefound to require the presence of HLA-A2.

Segregation studies demonstrated that each of mHag HA-1 to HA-5 is theproduct of a single gene segregating in a Mendelian fashion and thatHA-1 and HA-2 are not coded within the HLA region. The mHag differ fromeach other in phenotype frequencies: mHag HA-1 appeared relativelyfrequent (i.e., 69%) whereas mHag HA-2 appeared very frequent (i.e.,95%) in the HLA-A2-positive healthy population. An inventory in fivepatients of mHag HA-1, -2, -3, -4 and -5-specific anti-host CTLresponses after BMT demonstrated in three patients clones specific forthe mHag HA-1. This observation points towards the immunodominantbehavior of mHag HA-1. With regard to the mHag expressed on differenttissues, ubiquitous versus restricted tissue distribution of the mHaganalyzed were observed.

The expression of the mHag HA-1 was observed to be restricted to thecells of the hematopoietic cell lineage, such as thymocytes, peripheralblood lymphocytes, B-cells, and monocytes. Also the bone marrow-derivedprofessional APCs: the dendritic cells and the epidermal Langerhanscells were found to express the mHag HA-1. The mHag HA-1 was also foundto be expressed on clonogenic leukemic precursor cells, as well as onfreshly isolated myeloid and lymphoid leukemic cells, indicating thatmHag-specific CTLs are capable of HLA class-I restrictedantigen-specific lysis of leukemic cells.

To substantiate the importance of the human mHag systems, weinvestigated whether the mHag are conserved in evolution between humanand nonhuman primates. Hereto, cells from nonhuman primates weretransfected with the human HLAA2.1 gene. Subsequent analyses with ourhuman allo HLA-A2.1 and four mHag A2.1 restricted CTL clones revealedthe presentation of ape and monkey allo and mHag HY, HA-1 and HA-2peptides in the context of the transfected human HLA-A2.1 molecule byape and monkey target cells. This implicates that the HA-1 peptide isconserved for at least 35 million years.

A prospective study was performed in order to document the effect andclinical relevance of mHag in HLA genotypically identical BMT on theoccurrence of acute (grade≧2) GvHD. The results of the mHag typing usingthe CTL clones specific for five well-defined mHag HA-1 to HA-5demonstrated a significant correlation between mHag HA-1, -2, -4 and -5mismatch and GvHD. A significant correlation (P=0.024) with thedevelopment of GvHD was observed when analyzed for only mHag HA-1.

To analyze a putative peptidic nature of the mHag HA-1, we analyzed therequirement of the MHC encoded TAP1 and TAP2 gene products for mHagpeptide presentation on the cell surface. The transporter genes TAP1 andTAP2 associated with antigen presentation are required for delivery ofpeptides from the cytosol with the endoplasmic reticulum. Theavailability of a human cell line “T2” lacking both transport andproteasome subunit genes enabled us to study the processing andpresentation of human mHag. We demonstrated that the (rat) transportgene products TAP1 and TAP2u were required for processing andpresentation of antigenic peptides from the intracellular mH proteinHA-1. Information on the TCR repertoire post-BMT in man is extremelyscarce. We have analyzed the composition of the T-cell receptor (TCR) Vregion of mHag HA-1-specific CD8+ CTL clones by DNA sequencing of the αand β chains. We observed by analyzing TCR usage of 12 clones derivedfrom three unrelated individuals that the TcRβ chains all used theTCRβV6S9 gene segment and showed remarkable similarities within theN-D-N regions.

DISCLOSURE OF THE INVENTION

However, until the invention hereof, no one has succeeded in identifyingamino acid sequences of antigenic peptides relevant to the mHag HA-1antigen, nor has anyone succeeded in the identification of the proteinsfrom which this antigen is derived. We have now for the first timeidentified a peptide which is a relevant part of mHag HA-1.

Provided is a (poly)peptide comprising a T-cell epitope obtainable fromthe mHag HA-1 comprising the sequence VLXDDLLEA (SEQ ID NO:1) or aderivative thereof having similar functional or immunologicalproperties, wherein X represents a histidine (H) or an arginine (R)residue.

The way these sequences are obtained is further described herein. Animportant part of this method of arriving at the sequences is thepurification and the choice of the starting material. The method is,therefore, also part of the scope of this invention. However, now thatthe sequence is known, it is of course no longer necessary to followthat method, because the peptides can easily be made synthetically, asis well known in the art. Since routine techniques are available forproducing synthetic peptides, it is also within the ordinary skill ofone in the art to arrive at analogs or derivatives of the explicitlydescribed peptides, which analogs and/or derivatives may have the sameor at least similar functional or immunological properties and oractivity. Analogs which counteract the activity of the explicitlydescribed peptides are also within the skill of one in the art, giventhe teaching of the invention. Therefore, derivatives and/or analogs, bethey of the same or different length, be they agonist or antagonist, bethey peptide-like or peptidomimetic, are part of the scope of thisinvention.

The invention provides a (poly)peptide which can be functionallypresented to the immune system in the context of the HLA-A2.1 molecule.In general, peptides presented in such a context vary in length fromabout 7 to about 15 amino acid residues, and a polypeptide can beenzymatically processed to a peptide of such length. A peptide providedby the invention typically is at least 7 amino acids in length butpreferably at least eight or nine amino acids. The upper length of apeptide provided by the invention is no more than about 15 amino acids,but preferably no more than about thirteen, preferably eleven aminoacids in length. A peptide provided by the invention contains thenecessary anchoring residues for presentation in the groove of theHLA-A2.1 molecule. An immunogenic polypeptide provided by the inventioncomprises a 7 to 15 amino acid long peptide, provided by the invention,optionally flanked by appropriate enzymatic cleavage sites allowingprocessing of the polypeptide.

A preferred embodiment of the invention is a peptide of sequenceVLHDDLLEA (SEQ ID NO:2) that induces lysis of the cell presenting it ata very low concentration of peptide present. This does not imply thatpeptides inducing lysis at higher concentrations are not suitable. Thiswill for a large part depend on the application and on other propertiesof the peptides, which were not all testable within the scope of theinvention.

The peptides and other molecules of the invention are useful to inducetolerance of the donor immune system in HA-1-negative donors, so thatresidual peripheral blood lymphocytes in the eventually transplantedorgan or the bone marrow, as it may be, do not respond to host HA-1material in an HA-1-positive recipient. In this way, GvHD will beprevented or mitigated. On the other hand, tolerance is induced inHA-1-negative recipients in basically the same way, so that upon receiptof an organ or bone marrow from an HA-1-positive donor no rejection onthe basis of the HA-1 material occurs. For tolerance induction, verysmall doses can be given repeatedly, for instance, intravenously, butother routes of administration may very well be suitable too. Anotherpossibility is the repeated oral administration of high doses of thepeptides. The peptides may be given alone, or in combination with otherpeptides, or as part of larger molecules, or coupled to carriermaterials in any suitable excipients. Further applications of thepeptide or derivatives thereof lie in the prophylactic administration ofsuch to transplanted individuals to prevent GvHD. This can be done witheither agonists, possibly in combination with an adjuvant, or withantagonists which block the responsible cells. This can be done with orwithout the concomitant administration of TCR-derived peptide sequencesor of cytokines. Furthermore, the peptides of the invention are used toprepare therapeutic agents capable of eliminating a subset of cells,directly or indirectly, especially cells of hematopoietic origin and/ortumor cells. This can be illustrated by the following examples, whichrefer to leukemia related therapeutic agents.

An HA-1-positive recipient (in bone marrow transplantation) can besubjected to an additional pre-bone marrow transplant conditioningregime. This means that an agent which specifically recognizes a peptideof the invention (an HA-1 peptide) as presented selectively onhematopoietic cells and/or tumor cells, which agent induces eliminationof the cells presenting the peptide, is administered to the recipientbefore transplantation. This agent eliminates essentially all residualcells (leukemic cells) of hematopoietic origin as well asnon-hematopoietic tumor cells, if present. Such agents include but arenot limited to T-cells (which are tailor made ex vivo by pulsing withthe peptides provided by the invention, and optionally provided with asuicide gene) and/or antibodies coupled to toxic moieties.

An HA-1-negative donor for bone marrow transplantation can be vaccinatedwith a peptide of the invention, i.e., an HA-1 peptide. Upontransplantation to an HA-1-positive recipient, the donor's immune systemeliminates residual or recurrent HA-1 peptide-presenting cells in therecipient, which are, of course, leukemic or non-hematopoietic tumorcells. This is another example of tailor-made adoptive immunotherapyprovided herein.

A transplanted HA-1-positive recipient, transplanted with HA-1-negative(or for that matter HA-1-positive) bone marrow and suffering fromrecurrent disease (relapse), i.e., HA-1-positive leukemic cells, istreated with (again) an agent (as above) which specifically recognizes apeptide of the invention (an HA-1 peptide) as presented on hematopoieticcells, which agent induces elimination of the cells presenting thepeptide. In case of HA-1-positive bone marrow being transplanted to theHA-1-positive recipient, it is still very important (in case ofrecurrent disease) to eliminate all HA-1-positive cells even though thisincludes the transplanted material, because otherwise the HA-1-positiveleukemia will kill the recipient. To avoid the latter case, the patientcan be re-transplanted if necessary.

In such therapy protocols, one may first employ adoptive immunotherapywith agents (cells, antibodies, etc.) which specifically recognize andeliminate specific peptide expressing cells (e.g., leukemic cells) thatneed to be destroyed, after which in a second phase the patient isreconstituted with BMT cells replacing the killed cells. The inventionthus provides additional (or even substituting) protocols to othertherapeutic measures such as radiation.

Other therapeutic applications of the peptide include the induction oftolerance to HA-1 proteins in HA-1 related (auto)immune diseases. On theother hand they are used in vaccines in HA-1 related (auto)immunediseases.

Diagnostic applications are within the skill of the art. They include,but are not limited to HA-1 typing, detection of genetic aberrances andthe like. Specific gene sequences can be detected with various methodsknown in the art, such as hybridization or amplification with PCR andthe like. Immunological detection of peptides has also widely beenpracticed.

On the basis of the peptide described herein, genetic probes or primersare produced which can be used to screen for the gene encoding theprotein. On the other hand such probes are useful in detection kits aswell. On the basis of the peptide described herein, anti-idiotypicB-cells and/or T-cells and antibodies are produced. Various techniques,to allow detection of suitable donors or recipients, may be used, basedon amplification of HA-1 related nucleic acid sequences or on theimmunological detection of HA-1 related peptide sequences. Suitableamplification or detection techniques are known in the art, and theinvention enables the production of diagnostic test kits for HA-1allelic detection and typing. The GvHD-associated mHag HA-1 is a peptidederived from one protein allele of a di-allelic genetic system. Theidentification of this mHag HA-1 enables prospective HA-1 typing of BMTdonors and recipients to improve donor selection and thereby preventionof GvHD induction. All of these embodiments have been made possible bythe present disclosure and, therefore, are part of the invention. Thetechniques to produce these embodiments are all within the skill of theart.

Furthermore, the identification of the HA-1 antigen allows theproduction of synthetic HA-1 peptides and peptides functionally and/orimmunologically related thereto. Such peptides (which can include leftor right turning residues) can be designed and/or generated by variousmethods known in the art such as peptide synthesis and replacementmapping, followed by functional binding studies. Altered peptide ligands(APL) for the HLA-A2.1 restricted HA-1 epitope enable modification ofthe HA-1 directed T-cell responses and thus modulate and/or mitigate theGvHD-associated T-cell response. In general, T-cells are activated bythe interaction of the T-cell receptor (TCR) with the antigenic peptidein the context of a MHC molecule and can react with a number ofdifferent effector functions. APL can interact with the TCR and changethe effector functions of the T-cell qualitatively and/orquantitatively. APL, used in vitro as well as ex vivo can act asantagonist or agonist for the TCR and can energize T-cells specific forthe wild-type peptide. An HA-1 peptide is used to induce tolerance inthe living bone marrow or organ (kidney, liver, gut, skin, etc.) ofHA-1-(−) donors for HA-1-(+) patients. In bone marrow transplantation,the peptide (given alone or in combination with others) is used toinduce tolerance in the living bone marrow donor. The peptide(s) may begiven orally, intravenously, intra-occularly, intranasally or otherwise.In all forms of organ, tissue, and bone marrow transplantation, the HA-1peptide is used to induce tolerance in HA-1-negative recipients.

The invention also provides an analog of the peptide of the inventionantagonistic for the activity of T-cells recognizing the peptide. Afterbeing apprised of the disclosure given herein, such an analog may beobtained using methods and tests known in the art. Furthermore, theinvention provides a method for the generation of antibodies, T-cellreceptors, anti-idiotypic B-cells or T-cells, comprising the step ofimmunization of a mammal with a peptide or a polypeptide of theinvention, and the antibodies, T-cell receptors, B-cells or T-cellsobtainable by the method.

Dose ranges of peptides and antibodies and/or other molecules of theinvention to be used in the therapeutic applications as described hereinbefore are designed on the basis of rising dose studies in clinicaltrials for which rigorous protocol requirements exist.

An important advantage of using mHag-specific CTLs in adoptiveimmunotherapy of, for example, leukemia lies in their restricted andspecific target cell damage. We take advantage of three of the knowncharacteristics of human mHag, i.e., 1) MHC-restricted recognition byT-cells; 2) variable phenotype frequencies, i.e., mHag polymorphism; and3) restricted tissue distribution, allowing specific and distincttargeting of mHag HA-1 related therapy. Restrictive HA-1 tissueexpression significantly increases the success of adoptive immunotherapytowards various types of cancer, such as small cell lung carcinoma cellswhich express also the HA-1 antigen. Moreover, since mHag are clearlyexpressed on circulating leukemic cells and clonogenic leukemicprecursor cells of both myeloid and lymphoid origin, both types ofleukemias can be targeted. mHag peptide CTLs can be generated ex vivofrom mHag-negative BM donors for mHag-positive patients.Peptide-specific CTL clones from an HLA-A1-positive mHag-negativehealthy blood donor are generated by pulsing autologous APCs with mHagHA-1 related synthetic peptide. Proliferating clones are expanded andtested for specific cytotoxic activity. Upon transfusion (either pre-BMTas part of the conditioning regimen or post-BMT as adjuvant therapy),the mHag peptide-specific CTLs will eliminate the mHag-positivepatient's leukemic cells and, if of the patient's origin, also thepatient's hematopoietic cells but will spare the patient'snon-hematopoietic cells. If necessary, subsequent mHag-negative donorBMT will restore the patient's hematopoietic system. A universalapproach is to generate “prefab” mHag peptide-specific CTLs by usingmHag-negative healthy blood donors with frequent HLA-homozygoushaplotypes. Patients who are mHag-positive (and their BM donor'smHag-negative) and who match the HLA typing of the CTL donor can betreated with these “ready to be used” allo-peptide-specific CTLs.Transduction of these CTLs with a suicide gene allows elimination of theCTLs in case adverse effects occur.

Provided are means and methods for eliminating a tumor cell. HA-1 isalso present in tumor cells not of hematopoietic origin. HA-1 RNAtranscription was demonstrated by the present inventors in cell linesthat were generated from a wide array of tumors, i.e., breast,melanomas, lung, renal cell and colon carcinomas, hepatomas and head andneck cancers. Of these cell lines, the cell lines expressing HLA-A2 andHA-1 phenotypes, were lysed by HLA-A2 restricted HA-1-specific CTL. Thislysis demonstrates that HA-1 is indeed expressed in a functional way inthe tested cells.

HA-1 is not only expressed by tumor cell lines in vitro, which are proneto mutations, but also by tumor cells in vivo. The inventors verified byRNA analysis that also primary cancer cells express HA-1. Alsodisseminated cancer cells express HA-1. Disseminated cells from six of15 patients were found to be positive for HA-1. Expression of HA-1 isnot limited to a certain type of tumor cell. HA-1 expression was foundon different types of carcinoma cells in the patient population.

The observation that HA-1 is expressed in a functional way on themembrane of tumor cells of non-hematopoietic origin opens the road tomany different applications. One application is the use of HA-1expression on the tumor cell to target therapy to that cell. A bindingmoiety capable of specifically recognizing HA-1 is used to bind andeliminate the tumor cell. One aspect hereof, therefore, provides amethod for eliminating a tumor cell presenting an HA-1 mHag in thecontext of HLA class-I, wherein the elimination is induced directly orindirectly by specific recognition of the mHag in the context, themethod characterized in that the aberrant cell comprises anon-hematopoietic tumor cell that expresses HA-1. Preferably, the tumoris an epithelial tumor cell.

Several ways exist to induce elimination of a cell through specificrecognition of a target on that cell. In the invention emphasis is puton specific recognition by T-cells, however, the invention is notlimited to T-cells. Targeting is also possible with other bindingmolecules. By a “binding molecule” is meant herein any molecule orcompound (such as, for instance, a cell or at least part of an antibody)capable of binding an HA-1 epitope. The HA-1 epitope may be presented inthe context of MHC, but this is not necessary. The HA-1 epitope may, forinstance, alternatively be present in the context of the normal protein.Any type of binding molecule capable of specifically recognizing themHag in the context is suitable, provided that the molecule can mediateelimination of the cell, either directly (by means of a toxic effect) orindirectly, for instance, through binding of another compound thatcomprises a toxic effect. Such other toxic compound, for instance,comprises a cytostaticum. In one embodiment, the elimination is achievedthrough specific recognition by a murine or human(ized) antibodyspecific for HA-1 or specific for HA-1 presented in the context of MHC.Humanized or human monoclonal antibodies (though with differentspecificities) are used in, or developed for, a great variety ofanti-tumor therapies in the clinic.

A preferred means for inducing elimination of HA-1 expressing tumorcells comprises elimination induced by a T-cell comprising a T-cellreceptor specific for HA-1 presented in the context of MHC class-I. Thistechnology ties in with strategies for adoptive immunotherapy forhematopoietic malignancies.⁵⁶ Malignant cells derived from thehematopoietic system can express HA-1 and, therefore, form a target forT-cells comprising a specificity for HA-1 presented in the context ofMHC class-I.

With the teaching of the invention it is possible to extend theseapproaches to any type of tumor cell of non-hematopoietic origin. Theadoptive immunotherapy methods for hematopoietic malignancies are,therefore, also part of the invention and are incorporated herein byreference.⁵⁶ The cell directly involved in killing of these adoptiveimmunotherapy approaches is a cytotoxic T-cell. The invention thus alsoprovides a method for killing a human cell functionally expressing anHA-1 mHag in the context of HLA class-I, comprising incubating the cellwith a cytotoxic T-lymphocyte (CTL) specific for the mHag presented inthe context or incubating the cell with a functional equivalent of theCTL, the method characterized in that the human cell comprises anon-hematopoietic tumor cell.

A CTL specific for HA-1 in the context of HLA class-I is also used fordetermining whether a cell expresses functional levels of HA-1 in thecontext of HLA Class-I. For instance, (tumor) cells obtained from anindividual are screened for HA-1 expression to determine whether theindividual is HA-1-positive. The invention, therefore, further providesa method for determining whether a cell expresses functional levels ofan HA-1 mHag in the context of HLA class-I, comprising incubating thecell with a cytotoxic T-lymphocyte (CTL) specific for the HA-1 mHagpresented in the context and determining whether the cell and/or the CTLis affected. There are several ways to determine whether the cell or theCTL is specifically affected by the incubation. One typically usestarget cell killing to determine specific recognition by CTL, however,detection of gene expression characteristic for CTL mediated lysis inthe CTL or target cell can also, for instance, be used.

Now that we have demonstrated that HA-1 is expressed innon-hematopoietic tumor cells, the cells can be detected anddiscriminated from normal cells using methods for specifically detectingHA-1 in a cell. Typically, though not necessarily, the detection methodsutilize binding molecules capable of binding specifically to HA-1 and/ornucleic acid encoding the HA-1. For detection it is not required thatHA-1 is presented in the context of MHC class-I. Indeed preferably, theHA-1 or HA-1 encoding nucleic acid is present in the context of thenormal protein/gene. The invention, therefore, further provides a methodfor marking a non-hematopoietic tumor cell comprising incubating thecell with a molecule capable of specifically binding to an HA-1 mHagpresented in the context of HLA class-I, or capable of specificallybinding to a nucleic acid encoding the HA-1 mHag.

In principle, any type of method for specifically determining thepresence of a particular expression product is suitable for theinvention and is provided herewith. The HA-1 binding molecule may, forinstance, be labeled, such as with green fluorescent protein or aradioactive label. A cell which is bound to an HA-1 binding molecule canalso be detected with ELISA, affinity chromatography, etc. Of course,also provided is a non-hematopoietic tumor cell comprising a moleculecapable of specifically binding to an HA-1 mHag presented in the contextof HLA class-I, or capable of specifically binding to a nucleic acidencoding the HA-1 mHag.

Methods of the invention can be performed in vitro, however, in apreferred embodiment, a method is performed in vivo. In vivo, a methodof the invention can be used for the prevention and/or treatment ofdiseases caused by tumor cells. Provided is a method for at least inpart inhibiting expansion of a tumor in an individual comprisingproviding the individual with a CTL specific for an HA-1 mHag presentedin the context of HLA class-I, or a functional equivalent of the CTL,the method characterized in that the tumor cell comprises anon-hematopoietic tumor cell presenting the HA-1 mHag in the context ofthe HLA class-I. To obtain inhibition of expansion or even a reductionin tumor mass it is not required that all of the tumor cells expressHA-1. Though non-HA-1 expressing cells are not eliminated by a method ofthe invention, the removal of HA-1 expressing cells can still berelevant for treatment. Thus, inhibition of expansion of tumor cells canalso be achieved when only a part of the tumor cells express HA-1. Amethod of the invention may be performed in combination with other meansof tumor cell removal. In a preferred embodiment, the method is used tocombat recurrence of tumor in situations of minimal residual disease. Ofrelevance for in vivo applications is the fact that normal hematopoieticcells also express HA-1. Thus, any method capable of specificallyeliminating cells presenting HA-1 in the context of MHC class-I will, invivo, also affect a hematopoietic system. To this end it is preferred toprovide the individual with hematopoietic cells that are resistant tolysis by the CTL. This can be achieved in several ways. Preferably, anindividual is transplanted with hematopoietic stem cells comprising adifferent or no HA-1 and/or different MHC class-I alleles. In apreferred embodiment, the individual is transplanted with hemopoieticcells from an HA-1-negative donor. These cells cannot be recognized byHA-1-specific CTL and, thus, cannot be lysed by the T-cells. In apreferred embodiment, an individual is provided with stem cells that arenegative for HA-1 or comprise a different HA-1 but the same MHC class-Icompared to the tumor cell.

Methods for eliminating or killing tumor cells are suited for thetreatment of metastases, especially in the form of minimal residualdisease, in particular liver metastases.

Cells for transplantation may be obtained directly from a donor.However, current culture technology allows significant expansion ofcells in vitro, without detrimental effects on fitness and integrity ofthe cultured cells. Thus, cells for transplantation and T-cells can becultured in vitro if need be. One may also use T-cells that are educatedex vivo to comprise T-cell receptors capable of binding to HA-1presented in the context of MHC class-I. These educated T-cells can beexpanded further ex vivo before providing them to an individual. A fullyex vivo approach toward education and expansion of the right T-cells hasthe advantage that the cells can be analyzed and safety testedextensively before transplantation. A CTL of the invention is, forinstance, generated by incubating T-cells with dendritic cellscomprising the MHC class-I and the HA-1. The dendritic cells areprovided with the HA-1 by contacting with a peptide comprising the HA-1.Such systems also allow the generation of banks with extensivelycharacterized and tested T-cells with known specificities. Current andfuture technologies may be used to generate an HA-1-specific T-cell ofthe invention. For instance, current methods for the generation of suchT-cell include introduction of the genetic information for the relevantT-cell receptor into cells that are already T-cells or that can becomeT-cells. On the other hand functional equivalents of T-cells of theinvention are also possible. For instance, a suitable cloned T-cellreceptor is introduced in so-called empty cells that are T-cells fromwhich the relevant T-cell receptor is dysfunctional. Such T-cells do notexpress functional levels of native T-cell receptor chains and thus,cannot provide for chimeric T-cell receptors with unexpectedspecificities when provided with an HA-1-specific T-cell receptor.

In certain embodiments, T-cells and/or other hematopoietic cellsprovided to an individual comprise additional features. Such additionalfeatures can, e.g., be safety features, or additional (co)-stimulationfeatures. Safety can be built in, for instance, using so-called suicidegenes like Herpes Simplex Virus Thymidine Kinase (HSV-TK). Expression ofHSV-TK is toxic for many cells when a pro-drug like gancyclovir isprovided to the cells. A safety feature can be built in for a variety ofreasons, one of which is a relatively simple way to down-regulate thenumber of grafted cells in the body in case of undesired effects of thegrafted cells (GvHD and/or neoplasia). Additionally, other features canbe built in, like, for instance, features to improve the anti-tumoreffect of the grafted T-cells. This can be done by introducingco-stimulatory factors, cytokines and/or the encoding genes, therefore,into the T-cells. Thus, in certain embodiments, a method of theinvention is provided wherein the individual is provided with the CTL byproviding the individual with a graft comprising hematopoietic cells ofa donor.

In methods described herein, T-cells comprising a T-cell receptorspecific for HA-1 presented in the context of MHC class-I are beinggenerated, isolated, manipulated and/or provided with additionalfeatures. T-cells obtained with a method of the invention are,therefore, also part of the invention. With the current pace ofdevelopment in biological methods and knowledge, cells can be made intoantigen-specific T-cells in an artificial way, for instance, throughmanipulating programming genes. When such cells are made specific forHA-1 presented in the context of MHC class-I than such cells are T-cellsof the invention. T-cells of the invention and equivalents thereof canbe the basis for the preparation of medicaments for tumor cells. Theinvention thus also provides the use of an antigen-specific T-cell or anequivalent thereof having a specificity for HA-1 presented in thecontext of MHC class-I for the preparation of a medicament for treatinga disease related at least in part to non-hematopoietic tumor cells.Provided is also a use of a molecule capable of specifically binding anHA-1 mHag in the context of HLA class-I for preparing a medicament forthe treatment of disease related at least in part to non-hematopoietictumor cells. With a method of the invention, the growth of anon-hematopoietic tumor in an individual can be, at least in part,inhibited.

In an allogeneic SCT setting, donor lymphocyte infusion (DLI) therapyhas been clearly shown to be curative for hematologicalmalignancies.^(54, 55) However, DLI therapy is associated with GvHD. Totreat leukemia relapse after HLA matched, mHag HA-1 mismatched SCT withlow risk of GvHD, we have previously developed ex vivo protocols for thegeneration of donor-derived CTLs specific for the hematopoietic-specificmHag HA-1.⁵⁶ These SCT donor-derived HA-1-specific CTLs eliminate theHA-1-positive patient's hematopoietic and leukemic cells, whileHA-1-negative non-hematopoietic cells and tissues are spared. In the HLAidentical allogeneic SCT setting for solid tumors, GvT reactivity hasbeen demonstrated in small cohorts of patients with metastatic cancers,including breast cancer,^(46, 47, 57) melanomas,⁴⁸ renal cellcarcinomas⁴⁵ and ovarian carcinoma.⁴⁹ From the invention we know that atleast part of this GvT reactivity is due to tumor-specific polymorphicmHags such as HA-1. As in the leukemia transplant patients whereresidual leukemic tumor cells are present after high-dose chemotherapy,HA-1 directed immunotherapy is particularly warranted in cancer patientswith minimal residual tumor cells who were shown to confer an increasedrisk for a later occurring relapse.⁵⁸ Our observation of HA-1 expressionon various types of non-hematological tumor cells, offers a novel targetmolecule for therapy. Similar to the cellular immunotherapy protocol forthe treatment of relapsed leukemia, as described above, adoptiveimmunotherapy with donor-derived HA-1 CTLs in combination with SCT is anattractive alternative treatment of non-hematopoietic tumors,preferably, solid tumors. Because of the hematopoietic expression of theHA-1 gene, the patient's hematopoiesis will be at least partlyeliminated and needs reconstitution from donor SC or equivalentsthereof. Based on HA-1 restricted expression on malignantnon-hematopoietic tissues, HA-1 cellular therapy is specific, with noforeseen damage to normal tissues and cells. The above mentioned HA-1based immunotherapy capitalizes on expression of HLA-A2 and/or HLA-B60ligands for HA-1 CTL recognition, as is described below for HLA-B60, andthe HA-1^(H) allele of the HA-1 locus. Since there are many differentHLA-molecules it is expected that at least some other HLA molecules arealso capable of presenting HA-1. Methods of the invention are alsosuitable for these other HLA molecules. The HA-1^(H) phenotype frequencyis 69% in the HLA-A2-positive population.⁷ To our knowledge this is thefirst example of a constitutive hematopoietic-specific gene that canfunction as a universal tumor-specific antigen, with no significantexpression on its non-malignant counterparts. The significance of thepolymorphic mHag HA-1 for cancer therapy is underscored by the knownHA-1 immunogenic functional membrane expression and adequate CTLrecognition.

It may also be relevant to counteract an immune response against HA-1 inan individual. This may, for instance, be the case when the immuneresponse is no longer needed or when rescue of autologous cells isdesired, for instance, in case of inadvertent negative effects offoreign HA-1-specific T-cells. An infused cell can, for instance, becomeneoplastic, or otherwise deregulated in its cytokine excretion orresponsiveness. Thus, an efficient way of counteracting an immuneresponse against HA-1 is useful for a variety of reasons. Some ways tocounteract are given elsewhere in this application. Here another way ofcounteracting is provided. The invention in one aspect provides a methodto modulate mHag HA-1 response by providing an antagonistic peptide. Apeptide can be provided to an MHC class-I expressing cell in many ways,for instance, through providing the peptide, or the encoding nucleicacid. The peptide is designed such that it comprises a sufficientlystrong affinity for the HLA-molecule that HA-1 peptide is effectivelyprevented from associating with HLA, thereby at least in part reducingthe capability of the HA-1-specific immune response to attack the cell.

Also provided is a method for treating an individual suffering from orat risk of suffering from a non-hematopoietic tumor comprising inducingand/or enhancing in the individual an immune response against HA-1presented in the context of HLA class-I. In one embodiment, the immuneresponse is induced and/or enhanced by administering a CTL specific forHA-1 presented in the correct context. In another embodiment, the immuneresponse is induced and/or enhanced by vaccinating the individual with a(poly)peptide comprising HA-1 antigen. Vaccinations may be performedusing any method for vaccination against a peptide known in the art. Apreferred means of vaccination comprises the so-called string of beadsmethod of vaccination wherein several different peptides areincorporated into a proteinaceous molecule. When HA-1 antigen isprovided in the context of a larger molecule it is preferred that thepeptide comprising the HA-1 antigen is flanked at least on one side butpreferably on both sides by appropriate processing sites to allowcutting of the HA-1 antigen and the transport of the antigen to therelevant site and the association of the antigen with the appropriateMHC class-I molecule. Vaccinations can of course also be performed usingother methods known in the art. Such methods preferably comprise MHCtetramers. Vaccinations may be performed in the traditional sense orvaccination may be performed using artificial antigen-presentingmoieties in the form of liposomes comprising such MHC presenting therelevant HA-1 antigen. Vaccinations may of course comprise any suitabletype of adjuvant. Preferably, the adjuvant comprises CpG rich genes.This adjuvant is particularly preferred when vaccination is performedwith nucleic acid coding for an expressible HA-1 antigen.

In certain embodiments, an individual suffering from, or at risk ofsuffering from, a non-hematopoietic tumor is provided with hematopoieticstem cells from a donor, and is vaccinated with HA-1 antigen. The donorcells preferably express no, or a different, HA-1. Vaccination with HA-1at least partly solves the problem that allogeneic stem celltransplantation often results in GvH reactivity without tumorspecificity. Vaccination with HA-1 enhances the specificity of the GvHreaction to such an extent that GvHD is at least partly diminished. Incertain embodiments, a method is provided that involves a combination ofstem cell transplantation and HA-1 vaccination. Preferably, theindividual suffering from, or at risk of suffering from, anon-hematopoietic tumor is vaccinated. The vaccination is preferablyperformed after the individual has been provided with donor stem cells.It is, however, also possible to vaccinate a donor with HA-1 and tosubsequently provide an individual with donor hematopoietic cells,although vaccination of a healthy donor is usually not a method ofchoice. Various adjuvants can be used for the vaccination. For instance,donor dendritic cells comprising HA-1 can be used. Alternatively,GM-CSF, or CpGs, +HA-1 peptide can be used. In the art, many alternativeways of vaccination are known that can be used with a method of theinvention.

In certain embodiments, stem cell transplantation is performed bydirectly providing donor cells to the individual. Alternatively,adoptive immunotherapy can be used.

In general, peptides presented in the context of HLA vary in length fromabout 7 to about 15 amino acid residues, and a polypeptide can beenzymatically processed to a peptide of such length. A peptidecomprising HA-1 antigen provided by the invention typically is at least7 amino acids in length, but preferably at least 8 or 9 amino acids. Theupper length of a peptide provided by the invention is typically no morethan 15 amino acids, but preferably no more than about 11 to 13 aminoacids in length. A peptide provided herein contains anchoring residuesfor presentation in the groove of the relevant HLA molecule. Animmunogenic polypeptide provided by the invention comprises a 7 to 15amino acid long peptide, optionally flanked by appropriate enzymaticcleavage sites allowing processing of the polypeptide. Presentation ofthe HA-1 antigen by MHC class-I can occur in various ways depending onthe particular type of MHC class-I. Different HLA molecules behavedifferently in their capacity to present a peptide. In the inventionHA-1^(H) antigen can be presented by different HLA molecules. In case ofHLA-A2 the peptide presented comprises the sequence VLHDDLLEA (SEQ IDNO:2). When the HLA molecule is HLA-B60 the HA-1^(H) antigen comprises asequence that is shifted slightly when compared to the sequencepresented by HLA-A2. This is described in more detail below. However,the polymorphism is, of course, still present in the peptide presentedby HLA-B60. Thus, the HA-1 antigen may comprise any peptide capable ofbeing presented by an MHC class-I or for that matter MHC class-IImolecule provided that it comprises the relevant polymorphism.

In certain embodiments, the peptides and other molecules of theinvention find utility in that they induce and/or enhance an immuneinduced elimination of non-hematopoietic tumor cells. Since thehematopoietic cells of an HA-1-positive recipient also express HA-1, itis preferred that the individual having an immune response against HA-1in the context of HLA is induced and/or enhanced is provided withHA-1-negative hematopoietic stem cells. The HA-1 antigen containing(poly)peptides are used to prepare therapeutic agents capable ofeliminating a subset of cells, directly or indirectly, especially tumorcells of non-hematopoietic origin. This can be illustrated by thefollowing examples, which refer to leukemia related therapeutic agents.

An HA-1-positive, non-hematopoietic tumor baring recipient (in bonemarrow transplantation) is subjected to an additional pre-bone marrowtransplant conditioning regime. This means that an agent whichspecifically recognizes a (poly)peptide of the invention (an HA-1comprising (poly)peptide) as presented selectively on hematopoieticcells, which agent induces elimination of the cells presenting thepeptide, is administered to the recipient before transplantation. Thisagent will eliminate all (residual) tumor cells and cells ofhematopoietic origin. Such agents include but are not limited to T-cells(which are, e.g., tailor made ex vivo by pulsing with the peptidesprovided by the invention, and optionally provided with a suicide gene)and/or antibodies coupled to toxic moieties.

An HA-1-negative donor for bone marrow transplantation can be vaccinatedwith a peptide of the invention, an HA-1 peptide. Upon transplantationto an HA-1-positive recipient, the donor's immune system can eliminateany residual or recurrent HA-1 peptide-presenting cells in the recipientwhich are of course leukemic. This is another example of tailor-madeadoptive immunotherapy provided by the invention. A transplantedHA-1-positive recipient, transplanted with HA-1-negative (or for thatmatter HA-1-positive) bone marrow and suffering from recurrent disease(relapse), i.e., HA-1-positive tumor cells, can be treated with an agentas above which specifically recognizes a peptide of the invention (anHA-1 peptide) as presented on hematopoietic cells, which agent induceselimination of the cells presenting the peptide. In case ofHA-1-positive bone marrow being transplanted to an HA-1-positiverecipient, it is still very important (in case of recurrent disease) toeliminate all HA-1-positive cells even though this includes thetransplanted material, because otherwise the HA-1-positive tumor willkill the recipient. To avoid this, the patient can be re-transplanted,if necessary. In such therapy protocols it is possible to first employadoptive immunotherapy with agents (cells, antibodies, etc.) whichspecifically recognize and eliminate specific peptide expressing cells(e.g., tumor cells) that need to be destroyed, after which in a secondphase the patient is reconstituted with BMT cells replacing the killedcells. The invention thus provides additional (or even substituting)protocols to other therapeutic measures such as radiation.

A CTL capable of specifically killing a cell presenting HA-1 in thecontext of a suitable HLA class-I molecule is to be an HA-1-specificCTL, even in cases wherein the CTL was raised (educated) against adifferent peptide.

A (poly)peptide is to comprise an HA-1 antigen when a suitable part ofthe (poly)peptide is recognized by the aforementioned HA-1-specific CTLwhen the part is presented in the context of a suitable HLA molecule.

Now that the invention discloses that non-hematopoietic tumor cellsexpress HA-1, it is possible to use this information, e.g., indeveloping diagnostics. Considering that normal non-hematopoietic cellsdo not express HA-1, it is possible to discriminate between a tumornon-hematopoietic cell and a normal non-hematopoietic cell, on the basisof HA-1 gene expression. This can be done on the protein (peptide) leveland/or on the nucleic acid level. Also provided is a method for markinga non-hematopoietic tumor cell comprising incubating the cell with amolecule capable of specifically binding to an HA-1 mHag presented inthe context of HLA class-I, or capable of specifically binding to anucleic acid encoding the HA-1 mHag. Means and methods for determiningthe presence of HA-1 polypeptide or mRNA in a cell are well known to theperson skilled in the art. Examples of detection methods are describedin PCT International Publication WO 99/05313, which is hereinincorporated by reference. These methods may be combined with otherdetection and/or cell type characterization methods (for instance, forexpression products of other genes or microscopy) to exclude thepresence of HA-1 expressing hemopoietic cells. A cell comprising themolecule capable of specifically binding to an HA-1 mHag presented inthe context of HLA class-I, or capable of specifically binding to anucleic acid encoding the HA-1 mhag, is also part of the invention.

Also provided is a (poly)peptide that is functionally presented to theimmune system in the context of an HLA-A2.1 molecule. The (poly)peptideallows for HA-1-specific immunotherapy in patients positive forHLA-A2.1. However, a significant part of the population is negative forHLA-A2.1 and, hence, cannot be treated with the (poly)peptide or withbinding moieties recognizing the (poly)peptide in the context ofHLA-A2.1. A need exists for further HA-1 peptides able to associate withother HLA molecules. Particularly, a need exists for further HA-1peptides able to associate with other HLA class-I molecules. With suchHA-1 peptides, HA-1-specific immunotherapy can be extended to patientsthat are negative for HLA-A2.1.

We have found such further HA-1 peptides. A novel HLA-B60 restrictedT-cell epitope of HA-1 is provided comprising the sequence KECVLXDDL(SEQ ID NO:3). X represents either histidine or arginine. With theHLA-B60 restricted HA-1 epitope, it is possible to enlarge the patientpopulation for HA-1-specific immunotherapy. Patients that are negativefor HLA-A2.1 but positive for HLA-B60 can now be subjected to a methodof the invention.

In one aspect, the invention, therefore, provides a peptide constitutinga T-cell epitope obtainable from the mHag HA-1 comprising the sequenceKECVLXDDL (SEQ ID NO:3) or a derivative thereof having similarfunctional or immunological properties, wherein X represents a histidineor an arginine residue. The way these sequences are obtained isdescribed in the examples. Nonameric, as well as decameric, peptideshave been found that show strong binding capacity to HLA-B60 molecules.Especially, the nonameric and decameric HA-1^(H/R) peptides KECVLHDDL(SEQ ID NO:4), KECVLRDDL (SEQ ID NO:5), KECVLHDDLL (SEQ ID NO:6) andKECVLRDDLL (SEQ ID NO:7) show strong binding to HLA-B60 molecules.Hence, in certain embodiments, a peptide or a derivative of theinvention is provided, wherein the peptide comprises the sequenceKECVLXDDLL (SEQ ID NO:8). As used herein, an X in a sequence of apeptide of the invention represents a histidine or an arginine residue.

As described elsewhere herein, once a sequence of the invention isknown, peptides comprising such sequence can easily be madesynthetically. Methods for generating peptides synthetically are wellknown in the art. It is also within the skill of the art to arrive atanalogs or derivatives of a peptide of the invention. The analogs orderivatives can, for instance, be generated using conservativesubstitution. This means a substitution of one amino acid with anotheramino acid with generally similar properties (size, hydrophobicity,etc), such that the overall functioning is likely not to be seriouslyaffected.

The analog or derivative has the same or at least similar functional orimmunological properties and/or activity. “The same or at least similarfunctional or immunological properties and/or activity” means hereinthat at least one of the properties and/or activity is the same in kind,not necessarily in amount, as compared to the functional orimmunological properties and/or activity of the peptide the analog orderivative is derived from. Preferably, the analog or derivative hasessentially maintained most of the functional or immunologicalproperties and/or activity of the peptide in kind, not necessarily inamount. An analog or derivative of the above-mentioned HLA-B60restricted HA-1 epitope of the invention is preferably as well capableof binding an HLA-B60 molecule.

Provided is a (poly) peptide which can be functionally presented to theimmune system in the context of an HLA-B60 molecule. In general,peptides presented in such a context vary in length from about 7 toabout 15 amino acid residues, and a polypeptide can be enzymaticallyprocessed to a peptide of such length. A peptide provided by theinvention typically is at least 7 amino acids in length but preferablyat least 8 or 9 amino acids. The upper length of a peptide provided bythe invention is no more than about 15 amino acids, but preferably nomore than about 13 or 11 amino acids in length. A peptide provided bythe invention contains the necessary anchoring residues for presentationin the groove of the HLA-B60 molecule.

Thus, in one aspect, an immunogenic polypeptide obtainable from theminor Histocompatibility antigen HA-1 comprising the sequence KECVLXDDL(SEQ ID NO:3) or a derivative thereof having similar functional orimmunological properties is provided. In certain embodiments, thepolypeptide comprises the sequence KECVLXDDLL (SEQ ID NO:8).

An immunogenic polypeptide provided by the invention comprises a seven-to fifteen-amino acid long peptide of the invention, optionally flankedby appropriate enzymatic cleavage sites allowing processing of thepolypeptide.

In certain embodiments, a peptide comprising the sequence KECVLHDDL (SEQID NO:4) or KECVLHDDLL (SEQ ID NO:6) is provided, which induces lysis ofa cell presenting it at a very low concentration of peptide present.This does not imply that peptides inducing lysis at higherconcentrations are not suitable. This will for a large part depend onthe application and on other properties of the peptides, which were notall testable within the scope of the invention.

The peptides, derivatives and analogs of the invention find theirutility in that they are used to induce tolerance of the donor immunesystem in HA-1-negative donors, so that residual peripheral bloodlymphocytes in the eventually transplanted organ or the bone marrow, asit may be, do not respond to host HA-1 material in an HA-1-positiverecipient. In this way GvHD is prevented or mitigated. On the otherhand, tolerance is induced in HA-1-negative recipients in basically thesame way, so that upon receipt of an organ or bone marrow from anHA-1-positive donor no rejection on the basis of the HA-1 materialoccurs. For tolerance induction, very small doses are given repeatedly,for instance, intravenously, but other routes of administration may verywell be suitable too. Another possibility is the repeated oraladministration of high doses of the peptides. The peptides, derivativesand/or analogs thereof are given alone, in combination with otherpeptides, as part of larger molecules, or coupled to carrier materialsin any suitable excipients.

Further applications of a peptide, derivative and/or analog of theinvention lie in the prophylactic administration to transplantedindividuals to prevent GvHD. This is done with either agonists, possiblyin combination with an adjuvant, or with antagonists which block theresponsible cells. This can be done with or without the concomitantadministration of, for instance, TCR-derived peptide sequences orcytokines. Furthermore, a peptide, derivative and/or analog of theinvention is used to prepare a therapeutic agent capable of eliminatinga subset of cells, directly or indirectly, especially cells ofhematopoietic origin and/or tumor cells, as described above for HLA-A2.1restricted peptides of the invention comprising the sequence VLXDDLLEA(SEQ ID NO:1). The applications for an HLA-A2.1 restricted peptide ofthe invention, particularly suitable for HLA-A2.1-positive individuals,are similar to the applications of an HLA-B60 restricted peptide of theinvention which are particularly suitable for HLA-B60-positiveindividuals. HA-1-specific immunotherapy can now be applied toHLA-A2.1-positive, as well as HLA-B60-positive, patients.

As described elsewhere herein, aberrant cells of an HA-1-positivepatient, such as, for example, leukemic cells and/or tumor cells, areeliminated by administration to the patient of an agent specificallyrecognizing a peptide of the invention, the agent being capable ofinducing elimination of cells presenting the peptide. An HA-1-negativedonor for bone marrow transplantation is vaccinated with a peptide ofthe invention in order to enhance elimination by the donor's immunesystem of any HA-1 peptide-presenting cells in a recipient. Othertherapeutic applications of a peptide of the invention include inductionof tolerance to HA-1 proteins in HA-1 related (auto)immune diseases. Onthe other hand, they are used in vaccines, for instance, in HA-1 related(auto)immune diseases, tumor related diseases, etc.

Also provided is a pharmaceutical formulation and/or a vaccinecomprising a peptide or a polypeptide of the invention. A peptide orpolypeptide of the invention for use as a medicament is also herewithprovided. The medicament is preferably capable of inducing tolerance fortransplants to prevent rejection and/or GvHD. In another preferredembodiment, the medicament is capable of, at least in part, treating(auto)immune disease.

HA-1 is expressed in a functional way on the membrane of tumor cells,including tumor cells of non-hematopoietic origin. Hence, a peptidedescribed herein is also particularly suitable for inducing a (specific)immune response against a tumor. A use of a peptide or polypeptide ofthe invention in the preparation of a medicament for treatment of adisease that is at least in part related to tumor cells is, therefore,also provided. In a preferred embodiment, the tumor cells comprisenon-hematopoietic tumor cells.

Preferably, the peptide or polypeptide of the invention comprises apeptide constituting a T-cell epitope obtainable from HA-1 comprisingthe sequence KECVLXDDL (SEQ ID NO:3) or KECVLXDDLL (SEQ ID NO:8) or aderivative thereof having similar functional or immunologicalproperties.

Now that a peptide comprising an HLA-B60 restricted HA-1 epitope isprovided, one can, at least in part, eliminate a cell presenting thepeptide. The cell preferably comprises a hematopoietic cell (morepreferably a neoplastic hematopoietic cell) and/or a tumor cell (mostpreferably a non-hematopoietic tumor cell).

HA-1 was reported to be expressed in hematopoietic cells only. Of theinvention, however, HA-1 is as well expressed in a functional way on themembrane of tumor cells of non-hematopoietic origin. A binding moietycapable of specifically recognizing a cell presenting a peptide of theinvention is used to bind and eliminate the cell. One embodiment of theinvention, therefore, provides a method for the elimination of a(neoplastic) hematopoietic cell and/or a tumor cell presenting a peptideor polypeptide of the invention in the context of HLA-B60, wherebyelimination is induced directly or indirectly by specific recognition ofthe (poly)peptide in the context.

The binding moiety preferably comprises a cytotoxic T-lymphocyte. Asdescribed elsewhere, mHag peptide CTLs can be generated ex vivo frommHag-negative donors. Peptide-specific CTL clones from anHLA-B60-positive, HA-1-negative donor are, for instance, generated bypulsing autologous APCs with mHag HA-1 related synthetic peptide.Proliferating clones are expanded and tested for specific cytotoxicactivity.

In certain aspects, provided is a method for killing a hematopoieticcell and/or tumor cell functionally expressing an HA-1 mHag comprising apeptide or polypeptide of the invention in the context of HLA-B60,comprising incubating the cell with a cytotoxic T-lymphocyte (CTL)specific for the mHag presented in the context or incubating the cellwith a functional equivalent of the CTL. Preferably, the tumor cellcomprises a non-hematopoietic tumor cell. A CTL capable of specificallykilling a cell presenting HA-1 in the context of HLA-B60 is said to bespecific for an HA-1 presented in the context, even in cases wherein theCTL was raised (educated) against a different peptide.

Now that the invention discloses peptides comprising an HLA-B60restricted T-cell epitope of HA-1, it is possible to use the peptides indeveloping diagnostic tools. Since normal hematopoietic cells do notexpress HA-1, it is possible to discriminate between non-hematopoieticcells on the one hand and hematopoietic cells and/or tumor cells on theother. It is possible to discriminate between those cells on the basisof HA-1 gene expression. This can be done on the protein (peptide) leveland/or on the nucleic acid level. The invention, therefore, provides amethod for marking a hematopoietic cell and/or a tumor cell comprisingincubating the cell with a molecule capable of specifically binding toan HA-1 mHag comprising a peptide or polypeptide of the inventionpresented in the context of HLA-B60, or capable of specifically bindingto a nucleic acid encoding the HA-1 mHag. Preferably, the tumor cellcomprises a non-hematopoietic tumor cell.

As described above, means and methods for determining the presence ofHA-1 polypeptide or mRNA in a cell, such as, for instance, detectionmethods described in the incorporated PCT International publication WO99/05313, are well known to the person skilled in the art. These methodsmay be combined with other detection and/or cell type characterizationmethods (for instance, for expression products of other genes ormicroscopy) to exclude the presence of HA-1 expressing hematopoieticcells. A cell comprising the molecule capable of specifically binding toan HA-1 mHag presented in the context of HLA class-I, or capable ofspecifically binding to a nucleic acid encoding the HA-1 mHag, is alsopart of the invention.

It is often advantageous to test whether an individual expresses HA-1.For instance, a donor and recipient of bone marrow or organtransplantation are preferably tested in order to determine whether theyare mHag-matched. As another example, tumor cells obtained from anindividual can be screened for HA-1 expression. A CTL specific for HA-1in the context of HLA class-I can be used for determining whether a cellexpresses functional levels of HA-1 in the context of HLA Class-I. Inone embodiment of the invention, a CTL specific for a peptide of theinvention in the context of HLA-B60 is used. The invention, therefore,further provides a method for determining whether a cell expressesfunctional levels of an HA-1 mHag comprising a peptide or polypeptide ofthe invention in the context of HLA-B60, comprising incubating the cellwith a cytotoxic T-lymphocyte (CTL) specific for the HA-1 mHag presentedin the context and determining whether the cell and/or the CTL isaffected.

Several ways exist to determine whether the cell or the CTL isspecifically affected by the incubation. One typically uses target cellkilling to determine specific recognition by CTL. Other ways are,however, possible as well, such as detection of a gene expressionpattern characteristic for CTL mediated lysis in the CTL or target cell.

Also provided is an analog of a peptide hereof antagonistic for theactivity of T-cells recognizing the peptide (preferably in the contextof HLA-B60). Such an analog may be obtained using methods and testsknown in the art. Furthermore, provided is a method of generatingantibodies, T-cell receptors, anti-idiotypic B-cells or T-cells,comprising immunizing a (preferably HLA-B60-positive) mammal with a(poly)peptide hereof. Antibodies, T-cell receptors, B-cells or T-cellsobtainable by the method are also herewith provided. Dosage ranges ofpeptides, antibodies and/or other molecules hereof to be used intherapeutic applications are designed on the basis of rising dosestudies in clinical trials for which rigorous protocol requirementsexist.

As described elsewhere herein, T-cells can be educated in vivo as wellas ex vivo to comprise T-cell receptors capable of binding to a minorantigen presented in the context of MHC class-I. Now that a peptidecomprising an HLA-B60 restricted HA-1 epitope is provided herein,cytotoxic T-cells against the peptide/epitope are also generated. A CTLof the invention is generated by incubating a T-cell with anantigen-presenting cell (APC), preferably a dendritic cell, comprisingthe peptide. The APC most preferably comprises HLA-B60. The APCs may beprovided with an HA-1 epitope of the invention by contacting the cellswith a peptide comprising the HA-1 epitope.

In certain embodiments, provided is a method for generating a cytotoxicT-cell against a minor antigen, comprising contacting a hematopoieticcell with a peptide or a polypeptide of the invention. The minor antigenpreferably comprises HA-1. More preferably, the hematopoietic cell iscontacted with the peptide or polypeptide in the context of HLA-B60.

These educated T-cells can be expanded further ex vivo before providingthem to an individual. A fully ex vivo approach toward education andexpansion of the right T-cells has the advantage that the cells can beanalyzed and safety tested extensively before transplantation. Hence, aT-cell of the invention is preferably capable of expansion.

In a preferred embodiment, the hematopoietic cell itself is negative forthe minor antigen. This allows exposure of a T-cell to a peptide of theinvention without exposure to additional epitopes of the minor antigen.

As described elsewhere, current and future technologies may be used togenerate an HA-1-specific T-cell of the invention, and functionalequivalents of T-cells of the invention are also part of the invention.

The invention also includes a T-cell, or derivative or active fragmentthereof, specifically directed against a peptide of the invention. TheT-cell is preferably a cytotoxic T-cell. In one embodiment, the T-cell,derivative or active fragment may be obtained with a method of theinvention. Preferably, the T-cell, derivative or active fragment isspecifically directed against a peptide of the invention in the contextof an HLA-B60 molecule.

With the current pace of development in biological methods and knowledgecells can be made into antigen-specific T-cells in an artificial way,for instance, through manipulating programming genes. When such cellsare made specific for HA-1 presented in the context of MHC class-I thansuch cells are T-cells of the invention.

In one embodiment, T-cells of the invention are provided comprisingadditional features. Such additional features, for instance, comprisesafety features, or additional (co)-stimulation features. Safety is, forinstance, built in using so-called suicide genes. Additionally, otherfeatures can be built in, like, for instance, features to improveanti-tumor effect of grafted T-cells.

Once a CTL specific for HA-1 has been generated, it can be used tocounteract expansion of a tumor cell. Now that the invention providesmethods for generating CTLs specific for an HA-1 peptide in the contextof HLA-B60, tumor cells in HLA-B60-positive patients are counteracted aswell. As explained elsewhere, to obtain inhibition of expansion or evena reduction in tumor mass it is not required that all of the tumor cellsexpress HA-1. Inhibition of expansion of tumor cells can also beachieved when only a part of the tumor cells express HA-1.

One aspect of the invention, therefore, provides a method for at leastin part inhibiting expansion of a tumor cell in an individual comprisingproviding the individual with a CTL specific for an HA-1 mHag comprisinga peptide or polypeptide of the invention presented in the context ofHLA-B60, or a functional equivalent of the CTL. In a preferredembodiment, the individual is provided with the CTL by providing theindividual with a graft comprising hematopoietic cells of a donor.

A CTL specific for an HA-1 mHag can be generated ex vivo. However, theCTL can be generated in vivo as well. A peptide of the invention isadministered to an HLA-B60-positive individual that comprises a mismatchfor HA-1. In one application, the individual comprises a donor oflymphocytes. After generation of HA-1 directed T-lymphocytes, thelymphocytes can be used for counteracting a tumor, preferably anon-hematopoietic tumor, in an HA-1-positive patient. Thus provided is ause of an HA-1 antigen comprising a (poly)peptide hereof for inducingand/or enhancing the generation of HA-1-specific cytotoxic lymphocytesin an HA-1-negative donor of lymphocytes, wherein the generatedlymphocytes are used in a method for treating a disease that is at leastin part related to tumor cells.

Alternatively, A CTL specific for an HA-1 mHag can be generated in vivoby administration of a tumor cell expressing the HA-1 mHag in thecontext of HLA-B60 to an HLA-B60-positive individual that comprises amismatch for HA-1. Upon administration, the individual will produce HA-1directed CTL. A method of the invention is preferably performed in anon-human animal in order to produce the desired CTLs. Alternatively, amethod of the invention is used for vaccination purposes.

One embodiment of the invention thus provides a method for generating aCTL capable of binding to an HA-1 mHag comprising a peptide orpolypeptide of the invention, presented in the context of HLA-B60,comprising the step of administering to an individual that comprises amismatch for the HA-1 mHag, a tumor cell expressing the HA-1 mHagpresented in the context. The tumor cell preferably comprises anon-hematopoietic tumor cell.

A molecule capable of specifically binding an HA-1 mHag in the contextof HLA-B60, such as the above-mentioned CTLs, is particularly suitablefor eliminating tumor cells. Hence, a medicament is generated comprisingthe binding molecule. A use of a molecule capable of specificallybinding an HA-1 mHag comprising a peptide or polypeptide of theinvention in the context of HLA-B60 for the preparation of a medicamentfor the treatment of a disease that is at least in part related to tumorcells is, therefore, also provided herewith. Preferably, the tumor cellscomprise non-hematopoietic tumor cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Reconstitution of HA-1 with HPLC fractionated peptides elutedfrom HLA-A2.1 molecules in a ⁵¹Cr-release assay with mH HA-1-specificT-cell clone 3HA15. FIG. 1A: Peptides were eluted from 90.10⁹ HA-1- andHLA-A2.1-positive Rp cells and separated using reverse phase HPLC withHFBA as organic modifier. FIG. 1B: Fraction 24 of the first HPLCdimension that contained HA-1 activity was further fractionated byreverse phase HPLC with TFA as organic modifier. FIG. 1C: HA-1containing fraction 27 of the second gradient was furtherchromatographed with a third shallower gradient consisting of 0.1%acetonitrile/minute. Background lysis of T2 by the CTL in the absence ofany peptides was: in FIG. 1A, 3%; in FIGS. 1B and 1C, 0%. Positivecontrol lysis was: in FIG. 1A, 99%; in FIG. 1B, 74%; and in FIG. 1C,66%. FIG. 1D: Determination of candidate HA-1 peptides. HPLC fraction 33from the separation in FIG. 1C was chromatographed with an on-linemicrocapillary column effluent splitter and analyzed by electrosprayionization mass spectrometry and a ⁵¹Cr-release assay. HA-1reconstituting activity as percent specific release was compared withthe abundance of peptide candidates measured as ion current.

FIG. 2: Sequencing of mH HA-1 peptide by tandem mass spectrometry. FIG.1A: Collision activation dissociation mass spectrum of peptide candidate(SEQ ID NO:14) with m/z of 513. FIG. 1B: Reconstitution assay withdifferent concentrations of synthetic mH HA-1 peptide with threeHA-1-specific T-cell clones, 3HA15, clone 15 and 5W38. Background lysisof T2 by the CTL in the absence of any peptide was for 3HA15, 4%; forclone 15, 10%; and for 5W38, 2%. Positive control lysis was for 3HA15,46%; for clone 15, 47%; and 5W38, 48%.

FIG. 3: KIAA0223 polymorphism exactly correlated with mHag HA-1phenotype. FIG. 3A: The HA-1 region of KIAA0223 was sequenced in an HA-1mHag typed family. Six PCR products of each family member weresequenced. Family members 00, 07 and 09 expressed the HA-1^(R) in allsix PCR products. Family member 01 expressed the HA-1^(H) allele in twoPCR products and the HA-1^(R) allele in four PCR products. Family member02 expressed the HA-1^(H) allele in three PCR products and the HA-1^(R)allele in three PCR products. Family member 08 expressed the HA-1^(H)allele in four PCR products and the HA-1^(R) allele in two PCR products.FIG. 3B: HA-1 allele-specific PCR reaction in an HA-1 mHag typed familycorrelated exactly with the HA-1 phenotype. The sizes of the resultingPCR products were consistent with the expected sizes deduced from thecDNA sequence. FIG. 3C: Transfection of the HA-1^(H) allele of KIAA0223leads to recognition by mHag HA-1-specific T-cells. The HA-1^(H) and theHA-1^(R) coding sequence of KIAA0223 were together with HLA-A2.1transfected into Hela cells. After three days, the HA-1-specific CTLclones 5W38 and 3HA15 were added and after 24 hours, TNFα release wasmeasured in the supernatant. The clone Q66.9 is specific for theinfluenza matrix peptide 58-66. No TNFα production was observed aftertransfection of the pcDNA3.1(+) vector alone (results not shown).

FIG. 4: FIG. 4A: Binding of HA-1^(H) and HA-1^(R) peptides to HLA-A2.1.The binding of HA-1^(H) and HA-1^(R) peptides were assayed for theirability to inhibit the binding of fluorescent peptide FLPSDCFPSV (SEQ IDNO:9) to recombinant HLA-A2.1 and b2-microglobulin in a cell-freepeptide binding assay. One representative experiment is shown. The IC50is determined on the results of four experiments and was 30 nM forVLHDDLLEA (SEQ ID NO:2) and 365 nM for VLRDDLLEA (SEQ ID NO:10). Alsoshown is YIGEVLVSV (SEQ ID NO:77). FIG. 4B: Reconstitution assay withdifferent concentrations of synthetic HA-1^(R) peptide withHA-1-specific T-cells. The HA-1^(R) peptide was titrated andpreincubated with T2 cells. Three HA-1-specific T-cell clones, 5W38,3HA15 and clone 15 were added and a four-hour ⁵¹Cr-release assay wasperformed. Background lysis of T2 by the CTL in the absence of anypeptide was for 3HA15, 4%; for clone 15, 10%; and for 5W38, 2%. Positivecontrol lysis was for 3HA15 46%; for clone 15, 47%; and 5W38, 48%.

FIG. 5. Cytotoxic T-cell activity against peptide pulsed andmHag-positive target cells by two ex vivo induced HA-1 (FIGS. 5A, 5B)and two ex vivo induced HA-2-specific CTLs (FIGS. 5C, 5D). CTLs shown inFIGS. 5A, 5C, and 5D are induced using PBDC, whereas CTLs shown in FIG.5B are induced using BMDC. Target cells: autologous PHA blasts (⋄);autologous PHA blasts pulsed with peptide (♦); EBV-LCL positive for HA-1(n=4) or HA-2 (n=3) (▴); EBV-LCL negative for HA-1 (n=3) or HA-2 (n=3)(◯); HA-1- or HA-2-negative EBV-LCL pulsed with HA-1 or HA-2 peptide().

FIG. 6: Hematopoietic cell restricted cytolysis mediated by in vivo(FIGS. 6A, 6C) and ex vivo (FIGS. 6B, 6D) induced HA-1- (FIGS. 6A, 6B)and HA-2- (FIGS. 6C, 6D) specific CTLs. All target cells were derivedfrom the same HLA-A2+, HA-1+, HA-2+ blood donor. Target cells: PHAblasts (▴); fibroblasts (♦); Fibroblasts cultured with IFN-γ+TNF-α (both300 U/ml) (); Fibroblasts cultured with IFN-γ plus TNF-α and pulsedwith 10 μg/ml peptide (◯).

FIG. 7: Lysis of HA-1+ (FIGS. 7A, 7B, 7C) or HA-2+- (FIGS. 7D, 7E, 7F)positive leukemic cells by in vivo (FIGS. 7B, 7E) and ex vivo (FIGS. 7C,7F) induced HA-1- and HA-2-specific CTLs. Lysis of target cells bycontrol HLA-A2-specific CTL clone is shown in FIGS. 7A and 7D. Targetcells: HA-1- or HA-2-negative EBV-LCL (▴); HA-1- or HA-2-positiveEBV-LCL (Δ), Leukemic cells positive for HA-1 (n=4) or HA-2 (n=3) (♦);HA-1- or HA-2-positive leukemic cells cultured with IFN-γ+TNF-α ().

FIG. 8. HA-1 gene expression in hematopoietic and non-hematopoieticcells. The relative HA-1 gene expression levels were determined by acalibration function generated from RNA of the HA-1-positive KG-1 cellline. Cells of hematopoietic origin tested were: * PBMCs (n=3),

Dendritic cells (n=6), + Langerhans cells (n=2), □ EBV-LCLs (n=5),

PHA blasts (n=6),

Mast cell lines (n=3), X Monocytes (n=4), ◯ Thymocytes (n=3). Cells ofnon-hematopoietic origin tested were:

Keratinocytes (n=5), ▪ Fibroblasts (n=2),  PTECs (n=3),

HUVECs (n=3),

Melanocytes (n=3), two SV40 immortalized breast cell lines: ⋄ HaCat and♦ HBL-100.

FIG. 9: HA-1 and CD45 expression of micro-dissected tissue samples FIGS.9A and 9B. Micro-dissection of primary tumor (adenocarcinoma of thelung) and normal breast gland, respectively. FIG. 9C: Three to six areasfrom tumor samples (about 10,000 to 50,000 μm² of a 5 μm section) wereindividually analyzed by gene-specific PCR for HA-1 (primer HA-1 (II))and CD45. The same was done using pooled cDNA from several milk ducts(in total 60,000 μm² each) from normal breast tissue of three differentdonors (controls 1 to 3). Lane numbering indicates the differentmicro-dissected areas, PN=patient number. Arrows point to tumor areaswithout contaminating hematopoietic cells but positive HA-1 signal.

FIG. 10: Isolation and gene expression analysis of single disseminatedcancer cells or small tumor cell clusters. FIG. 10A: Three-cell cluster(PN5-C4) after micromanipulator-assisted isolation from a cellsuspension of a lymph node preparation. All cells of the cluster areintensively stained by the EpCAM antibody. FIG. 10B: Gene expressionprofiling on cDNA array of isolated tumor cells. HA-1 expression afterstandard RT-PCR is given in the first line. The grey shades representthe signal intensity (from light grey=weak signal to black=strongsignal).

FIG. 11: HA-1 expression of disseminated cancer cells. Cells positivefor the HA-1 (II) primer pair were digested with Hinf I, blotted andhybridized with the respective probe. M=size marker; A=undigested PCRproduct; B=Hinf I digested product; lane 1=PN12-C1; lane 2=PN4-C1; lane3=PN3-C1; lane 4=PN5-C1; lane 5=PN6-C5; lane 6=PN2-C1; lane+=HT29 forHA-1 and normal bone marrow for CD45.

FIG. 12: CGH profile of cell PN3-C1. Each chromosome is represented byits ideogram and numbered. Deletions are marked with a bar (e.g., lossof chr. 13) at the left and gains with a bar (e.g., gain of chr. 8q) atthe right side of the chromosome symbol.

FIG. 13: Binding of HA-1^(H/R) peptides to HLA-A3. The results areexpressed as the percentage inhibition of the HLA binding of the 150 nMfluorescent reference peptide by the indicated peptides VLHDDLLEAR (SEQID NO:43) and VLRDDLLEAR (SEQ ID NO:44) added at serial dilutions (seematerial and methods). Curves were fitted by nonlinear regression andone site binding equation. The IC₅₀ value of the HLA-A3 binder-positivecontrol peptide KQSSKALQR⁹ (SEQ ID NO:11) was 9.4 μM.

FIG. 14: Efficient binding of HA-1^(H/R) peptides to HLA-B60. Theresults are expressed as the percentage inhibition of the HLA binding ofthe 150 nM reference peptide by the indicated peptides KECVLHDDL (SEQ IDNO:4), KECVLRDDL (SEQ ID NO:5), KECVLHDDLL (SEQ ID NO:6), and KECVLRDDLL(SEQ ID NO:7) added at serial dilutions (see material and methods).Curves were fitted by nonlinear regression and one site bindingequation. The IC₅₀ value of the HLA-B60 binder-positive control peptideKESTLHLVL⁹ (SEQ ID NO:12) was 1.1 μM.

FIG. 15: Stable binding of nonameric and decameric HA-1^(H/R) peptidesto HLA-B60. The nonameric and decameric HA-1^(H/R) peptides were testedfor binding to HLA-B60 (FIGS. 15A, 15B) KECVLHDDLL (SEQ ID NO:6),KECVLHDDL (SEQ ID NO:4) and KECVLRDDLL (SEQ ID NO:7), KECVLRDDL (SEQ IDNO:5) and to HLA-A2 (FIG. 15C) VLHDDLLEA (SEQ ID NO:2) and VLRDDLLEA(SEQ ID NO:10) at the indicated temperatures. The results are expressedas the percentage inhibition of HLA the binding of the referencepeptide. Curves were fitted by nonlinear regression and one site bindingequation.

FIG. 16: T-cell recognition of HLA-B60/HA-1^(H) ligand. T-cell lines(TCL) secreting IFN-γ in response to the target cells indicated. TheEBV-LCLs (HLA-B60/HA-1^(RR) and HLA-B60/HA-1^(HR)) are derived from HLAidentical but HA-1 non-identical siblings. On the Y-axis, the number ofIFN-γ spots per 10⁵ cells is expressed. The SEM was <5%.

Table 6: KIAA0223 sequence polymorphism in mH HA-1-positive andHA-1-negative individuals. Sequencing of HA-1 region in KIAA0223 gene inHA-1+/+ and HA-1−/− homozygous individuals and KG-1 revealed two allelesdiffering in two nucleotides resulting in a one amino acid difference (Hto R) and designated HA-1^(H) and HA-1^(R). For DH and vR, sixindependent PCR products were sequenced. For KG-1, 8 PCR products weresequenced.

DETAILED DESCRIPTION OF THE INVENTION

For the sake of illustration, a number of methods and applications arealso given below in the examples.

EXAMPLES Example 1

GvHD is a frequent and life-threatening complication after allogeneicHLA-identical bone marrow transplantation (BMT). Recipients ofHLA-identical bone marrow develop acute or chronic GvHD in respectively36% and 49%.^(1, 2) Disparities in genes other than the MHC, referred toas minor histocompatibility antigens (mHags), are clearly involved inthe development of GvHD after HLA-identical BMT. A recent retrospectiveanalysis revealed the significant association between mismatching forthe mHag HA-1 and the induction of GvHD after HLA-identical BMT.³ mHagsare recognized by MHC restricted T-cells and were shown to be peptidesderived from intracellular proteins presented by MHC molecules.⁴⁻⁶ Herewe report the first identification of a polymorphic gene encoding anhuman mHag. The GvHD-associated mHag HA-1 is a nonapeptide derived fromthe di-allelic KIAA0223 gene. The HA-1 allelic counterpart encoded bythe KIAA0223 gene differs only at one amino acid from the mHag HA-1.Family studies demonstrated an exact correlation between the KIAA0223gene polymorphism and the HA-1 phenotype as was previously determined byrecognition by the HA-1-specific CTL clones. The elucidation of the HA-1encoding gene enables prospective HA-1 DNA typing of BMT donors andrecipients to improve donor selection and prevention of GvHD.

Cytotoxic T-cell clones specific for the mHag HA-1 have been isolatedfrom three different patients with severe GvHD.⁷ The inHag HA-1 ispresented in the context of HLA-A2.1 and present in 69% of theHLA-A2.1-positive population.⁷ HA-1 expression was demonstrated to betissue-specific and limited to cells of hematopoietic origin, includingdendritic cells, Langerhans cells and leukemic cells.⁸⁻¹⁰ Familyanalysis indicated a Mendelian mode of inheritance for HA-1 andsegregation independent from the MHC complex.¹¹ Comparison of the T-cellreceptor (TCR) sequences of different HA-1-specific T-cell clonesderived from different individuals revealed conserved usage of the TCRVβ6.9 and conserved amino acids in the CDR3 region.¹² In a retrospectivestudy, mismatching for a number of mHags was evaluated with regard tothe association with GvHD after HLA-identical BMT. A single HA-1mismatch between donor and recipient was significantly correlated withthe induction of GvHD after HLA-identical BMT.³

To identify the mHag HA-1, HLA-A2.1 molecules were purified from twoHA-1 expressing EBV-transformed B lymphoblastoid cell lines (EBV-BLCL)Rp and Blk. The HLA-A2.1 bound peptides were isolated by acid treatmentand fractionation of the peptides was performed by multiple rounds ofreverse phase HPLC. The fractions were analyzed for their capacity ofinducing HA-1-specific lysis using T2 cells as target cells and anHA-1-specific CTL clone as effector cells in a ⁵¹Cr-release assay (FIG.1A). Fraction 24 contained HA-1 activity and was two times furtherfractionated with reverse phase HPLC using a different organic modifier(FIGS. 1B and 1C). Fraction 33 and 34 of the third HPLC fractionationshowed HA-1 activity ⁵¹Cr-release assay and were analyzed by tandem massspectrometry. Because over 100 different peptides were present in thesefractions, around 40% of fractions 33 and 34 were chromatographed withan on-line microcapillary column effluent splitter. The fractions weresimultaneously analyzed by tandem mass spectrometry and ⁵¹Cr-releaseassay (FIG. 1D). Five peptide species (at m/z 550, 520, 513, 585 and502) were specifically present in active fractions and absent infractions without activity in the CML assay. Collision-activateddissociation analysis of peptide candidate m/z 550 revealed the sequenceYXTDRVMTV (SEQ ID NO:13). X stands for Isbleucine or leucine that cannotbe discriminated with this type of mass spectrometer. However, asynthetic peptide with this sequence was not able to reconstitute theHA-1 epitope (results not shown). To determine which of the fourremaining candidates was the HA-1 peptide the second HA-1 purificationof the EBV-BLCL Blk was evaluated. HA-1-positive peptide fraction 33 ofthe second reverse phase HPLC fractionation was further chromatographedby microcapillary HPLC with a third organic modifier. A single peak ofreconstituting activity was observed in a ⁵¹Cr-release assay (resultsnot shown). Mass spectral analysis of these fractions revealed that onlypeptide candidate m/z 513 was present. This peptide was analyzed withcollision-activated dissociation analysis and sequenced as VXHDDXXEA(SEQ ID NO:14) (FIG. 2A). Isoleucine and leucine variants of the peptidewere synthesized and run on the microcapillary HPLC column. Only peptideVLHDDLLEA (SEQ ID NO:2) coeluted with the naturally processed peptide513 (results not shown). Next, synthetic VLHDDLLEA (SEQ ID NO:2) addedin different concentration to a CML assay with three differentHA-1-specific CTL clones revealed recognition by all three clones of thepeptide with a half maximal activity at 150 to 200 pM for or all threeclones (FIG. 2B). This demonstrated that the mHag HA-1 is represented bythe nonapeptide VLHDDLLEA (SEQ ID NO:2).

Database searches performed to identify the gene encoding HA-1, revealedthat the HA-1 peptide VLHDLLEA (SEQ ID NO:15) was identical for eightout of nine amino acids with the peptide VLRDDLLEA (SEQ ID NO:10) fromthe KIAA0223 partial complementary DNA (CDNA) sequence, derived from theacute myelogenous leukemia KG-1 cell line. Because HA-1 has a populationfrequency of 69%, we reasoned that VLRDDLLEA (SEQ ID NO:10) mightrepresent the HA-1 allelic counterpart present in the remaining 31% ofthe population. To elaborate on this assumption, we performed cDNAsequence analysis of the putative HA-1 encoding region of KIAA0223 inEBV-BLCL derived from a presumed HA-1 homozygous-positive (vR), from apresumed HA-1-negative individual (DH) and from the KG-1 cell line(Table 6.). The HA-1 encoding region of KIAA0223 of the HA-1+/+individual (vR) displayed two nucleotides differences from the KIAA0223sequence in the databank, leading to the amnino acid sequence VLHDDLLEA(SEQ ID NO:2) (designated HA-1^(H)). The HA-1 encoding region ofKIAA0223 of the HA-1−/− individual (DH) showed 100% homology with thereported KIAA0223 sequence (designated HA-1^(R)). The KG-1 cell lineexpressed both KIAA0223 alleles. Because KG-1 does not express therestriction molecule HLA-A2.1 necessary for T-cell recognition, wetransfected KG-1 with HLA-A2.1 and used these cells as target cells in a⁵¹Cr-release assay with the HA-1-specific T-cell clone as effectorcells. According to the cDNA sequence analysis results, the KG-1 cellswere recognized by the HA-1-specific T-cell clone (data not shown). Thisresult suggested that the KIAA0223 gene forms a di-allelic system ofwhich the HA-1^(H) allele leads to recognition by the mHag HA-1-specificT-cell clones.

Two families, who were previously typed for HA-1 with HA-1-specific CTLwere studied on the cDNA level for their KIAA0223 polymorphism. Thefamily members of family 1 were screened for their KIAA0223 sequencepolymorphism by sequencing the HA-1 encoding sequence region. AllHA-1-negative members displayed the HA-1^(R) sequence, whereas allHA-1-positive members turned out to be heterozygous, thus carrying bothHA-1 alleles (FIG. 3A). We subsequently designed HA-1 allele-specificPCR primers to screen another family previously cellularly typed forHA-1. Both parents and one child were determined as heterozygous forHA-1, two HA-1-negative children homozygous for the HA-1^(R) allele andone child homozygous for the HA-1^(H) allele (FIG. 3B). The screening ofboth families showed an exact correlation of the HA-1 phenotype asdetermined by recognition by the HA-1-specific T-cell clones and theKIAA0223 gene polymorphism.

To definitely prove that the KIAA0223 gene encodes the mHag HA-1, theHA-1 encoding sequence region of KIAA0223 of both the HA-1^(H) and theHA-1^(R) alleles were cloned in a eukaryotic expression vector andtransiently transfected in HA-1-negative Hela cells in combination withHLA-A2.1. HA-1-specific T-cell recognition of these transfected Helacells was assayed using a TNFα release assay. The Hela cells transfectedwith the HA-1^(H) sequence containing vector were recognized by twoHA-1-specific T-cell clones (FIG. 3C). In contrast transfection of theHA-1^(R) sequence containing vector did not lead to recognition. Inconclusion, our results clearly demonstrate that the mHag HA-1 isencoded by the HA-1^(H) allele of the KIAA023 gene.

Reconstitution and HLA-A2.1 binding assays were performed to determinethe capacity of HA-1^(R) peptide VLRDDLLEA (SEQ ID NO:10) to bind toHLA-A2.1 and to be recognized by the HA-1-specific T-cell clones. Theconcentration of the HA-1^(R) peptide that inhibited the binding of afluorescent standard peptide to HLA-A2.1 by 50% (IC50) was 365 nM,falling in the intermediate binders, whereas the IC50 of the HA-1^(H)peptide was 30 nM, which is in the range of high affinity binders (FIG.4A).^(13, 14) Different concentrations of VLRDDLLEA (SEQ ID NO:10) weretested in a ⁵¹Cr-release assay with three HA-1-specific T-cell clones.One out of the three clones (3HA15) tested showed recognition of theHA-1^(R) peptide, but only at 1000 times higher peptide concentrationthan that necessary for the recognition of the HA-1^(H) peptide (FIG.4B). As the binding affinity of the two peptides to HLA-A2.1 differsonly ten-fold, it can be concluded that all the T-cell clonesspecifically recognize the HA-1^(H) peptide.

The 3HA15 T-cell clone, recognizing the HA-1^(R) peptide at highconcentrations, does not recognize HA-1^(R) homozygous individuals. Thissuggests that SEQ ID NO:10 is not presented by HLA-A2.1 or presentedbelow the detection limit of the T-cell. To determine whether theHA-1^(R) peptide SEQ ID NO:10 was presented by HLA-A2.1, HLA-A2.1 boundpeptides were eluted from an HA-1^(R) homozygous EBV-BLCL andfractionated with reverse phase HPLC. The synthetic HA-1 peptide SEQ IDNO:10 was run on reverse HPLC to determine at which fraction thispeptide eluted. The corresponding HPLC fractions derived from theHA-1^(R) expressing EBV-BLCL were analyzed using mass spectrometry.Presence of peptide SEQ ID NO:10 could not be detected (results notshown), indicating that this peptide is not, or is in very low amounts,presented by HLA-A2.1 on the cell surface. This is most likely due tothe ten-fold lower binding affinity of the peptide for HLA-A2.1. Thesupposed absence of the HA-1^(R) peptide in HLA-A2.1 indicates that thisallele must be considered as a null allele with regard to T-cellreactivity. This implicates that only BMT from an HA-1^(R/R) (HA-1-)donor to HA-1^(H/H) or HA-1^(R/H) (HA-1+) recipient direction and notthe reverse would be significantly associated with GvHD. This is indeedobserved in a retrospective study in which HLA-2.1-positive BMT pairswere typed for HA-1.³ However, HA-1^(R)-derived peptides may bind toother HLA alleles and possibly be recognized by T-cells. If the latterpeptides are not generated and presented by the HA-1^(H) allele, thenT-cell reactivity towards the HA-1^(R) allele may be envisaged and GvHDin that direction may occur.

Only a few murine and human mHags have been identified so far on thepeptide and gene level. Two murine mHags are encoded by mitochondrialproteins, leading to respectively four and two alleles.¹⁵⁻¹⁷ Inaddition, two murine H-Y mHags were shown to be peptides encoded byY-chromosome located genes.¹⁸⁻²¹ The human SMCY gene, located on the Ychromosome, encodes the HLA-B7 and the HLA-A2.1 restricted H-YmHags.^(5, 6) Of the human non-sex-linked mHags only the mHag HA-2 hasbeen sequenced on the peptide level, but the HA-2 encoding gene remainedunknown.⁴ The identification of the gene encoding the mHag HA-1 is thefirst demonstration that human mHags are derived from polymorphic genes.The HA-1 encoding KIAA0223 gene has two alleles differing in twonucleotides leading to one single amino acid difference. However,because the KIAA0223 gene has not been fully sequenced yet, it remainsto be established whether additional amino acid polymorphisms betweenthe two alleles of this gene are present.

Because the HA-1 mHag is the only known human mHag that is correlatedwith the development of GvHD after BMT the results of our study are ofsignificant clinical relevance.³ Although the numbers of different humanmHags is probably high, it is envisaged that only few immunodominantmHags can account for the risk for GvHD.²² Identification of those humanimmunodominant mHags and screening for those antigens may result in asignificant decrease in GvHD after BMT. Here, we describe the firstelucidation of a polymorphic gene encoding the immunodominant mfag HA-1.This enabled us to design HA-1 allele-specific PCR primers forpre-transplant donor and recipient typing to improve donor selection andthereby prevention of HA-1 induced GvHD development.

It also enabled us to start targeting leukemic cells carrying minorantigens present on hematopoietic cells. One way of arriving at agentstargeting leukemic cells, is the ex vivo preparation of CTLs. This isexplained herein below.

Allogeneic bone marrow transplantation (BMT) is a common treatment ofhematological malignancies.²⁹ Recurrence of the underlying malignancy isa major cause of treatment failure.^(30, 31) Relapsed CML patients canbe successfully treated by donor lymphocyte infusions (DLI),^(32, 33)but the treatment is less effective for relapsed AML and ALL,^(32, 33)and is frequently complicated with GvHD.³²⁻³⁴ Donor-derived CTLsspecific for patients' miHags play an important role in both GvHD andGvL reactivities.^(10, 35-38) mHags HA-1 and HA-2 induce HLA-A2restricted CTLs in vivo. mHags HA-1 and HA-2 are exclusively expressedon hematopoietic cells including leukemic cells^(10, 36) and leukemicprecursors,^(37, 38) but not on cells of the GvHD target organs such asskin fibroblasts, keratinocytes or liver cells.⁸ Recently the chemicalnature of the mHags HA-1 and HA-2 was unraveled.^(4, 39) Here we reporton the feasibility of ex vivo generation of mHag HA-1- and HA-2-specificCTLs from unprimed mHag HA-1- and/or HA-2-negative healthy blood donorswith the purpose of adoptive immunotherapy of relapsed leukemia with alow risk of GvHD.

To define the optimal APC for ex vivo generation of HA-1- andHA-2-specific CTLs, we prepared peripheral blood mononuclear cells(PBMC), monocytes, peripheral blood circulating dendritic cells (PBDC)or dendritic cells derived from bone marrow CD34+ progenitor cells(BMDC) from 15 HLA-A2-positive, HA-1- or HA-2-negative healthy blooddonors. These APCs were pulsed with HA-1 and/or HA-2 synthetic peptidesand used to stimulate autologous unprimed CD8⁺ T-cells. The attempts toinduce HA-1- or HA-2-specific CTLs using monocytes or PBMC were not verysuccessful. PBMC induced in only one out of three attempts HA-2-specificCTLs. Using monocytes, we generated two HA-1 peptide-specific CTLs, butthese CTLs did not lyse HA-1-positive target cells in our experiments(data not shown). It is possible that these “peptide-specific” CTLs havea lower affinity for the naturally expressed HA-1 antigen, but this doesnot mean that these cells cannot be used for generating CTLs againstminor antigens.

PBDC were enriched from nine individuals to induce HA-1- orHA-2-specific CTLs. In the four cases where the preparations had apurity of less than 30% the CTLs, lysed peptide loaded target cells, butnot mHag-positive target cells (data not shown). In contrast, in allcases (n=5) where PBDC purity was 30% or more, the CTLs not onlyrecognized mHag-negative, peptide pulsed target cells, but alsomHag-positive EBV-LCL, demonstrating the recognition of the naturallyexpressed ligand (FIG. 1). These results underscore the superiorcapacity of DC to induce T-cell responses from naive precursors andconfirm the current opinion.⁴⁰ Similarly, two BMDC induced CTLs thatrecognized both peptide pulsed target cells and HA-1-positive targetcells (FIG. 5). No cytotoxic activity was observed against autologousPHA stimulated T-cell blasts (PHA blasts) or against mHag-negativeEBV-LCL. Thus, neither autoreactivity nor “third-party” alloantigenreactivity was observed. Several HA-1- or HA-2-specific CTL clonesisolated from these CTLs did not react against autologous cells either.These results show that HA-1- and HA-2-specific CTLs can be safelytransferred to patients after BMT.

The ex vivo induced HA-1- and HA-2-specific CTLs were tested for theirhematopoietic cell restricted reactivity and compared with the in vivoinduced HA-1- and HA-2-specific CTLs (FIG. 6). PHA blasts, but notfibroblasts (even after IFN-γ/TNF-α stimulation) were recognized by bothex vivo and in vivo induced HA-1- and HA-2-specific CTLs. Fibroblasts,were only lysed after pulsing with the mHag peptides, demonstratingtheir susceptibility to CTL mediated lysis. These data not only confirmthat the HA-1 and HA-2 antigens are functionally expressed solely onhematopoietic cells,⁸ but also show that adoptive transfer of HA-1- orHA-2-specific CTLs to HA-1- or HA-2-positive patients spares thepatient's non-hematopoietic tissues and cells. Thus, upon adoptivetransfer of HA-1- and HA-2-specific CTLs, a low risk of GvHD is to beexpected. Some precaution may be necessary since we have previouslydemonstrated that HA-1 disparity between patient and donor is associatedwith the development of GvHD in adults.³ Therefore, we transfer the CTLsnot before 50 to 60 days post BMT. It is assumed that most recipienthematopoietic cells are then to be replaced by donor cells.Alternatively, one may transduce the HA-1- and HA-2-specific CTLs with asuicide gene which will make the in vivo elimination of cells possibleif adverse effects occur.⁴¹

The ex vivo induced HA-1- and HA-2-specific CTLs were subsequentlyanalyzed for cytolytic activity against, for this study the mostrelevant target cells, leukemic cells. In vivo induced HA-1- andHA-2-specific CTLs and an HLA-A2-specific alloreactive CTL were used ascontrol effector cells. As shown in FIG. 7, AML and ALL cells were lysedby HLA-A2-specific alloreactive CTL, and by in vivo induced HA-1- andHA-2-specific CTLs, indicating that the leukemic cells were positive forHLA-A2 and expressed HA-1 or HA-2 antigens. As expected the ex vivoinduced CTLs lysed the leukemic cells comparable to the control effectorcells. These results show that HA-1- and HA-2-specific CTLs can also beused as therapy for relapsed AML or ALL, which are resistant to DLItreatment. The level of cytotoxicity could be significantly enhancedfollowing IFN-γ and TNF-α treatment of the leukemic cells indicatingthat cytokines up-regulated HLA class-I expression on the leukemiccells. HA-1- and HA-2-specific CTL clones produce IFN-γ and TNF-α exvivo. It is possible that cytokine production by HA-1- and HA-2-specificCTLs occurs in vivo as well. Alternatively the efficacy of adoptiveimmunotherapy with HA-1- and HA-2-specific CTLs may be enhanced byco-administration of IFN-α in resistant cases.

The feasibility of adoptive immunotherapy with ex-vivo generated CTLsdepends on their expandability to sufficient numbers. We, therefore,scored the expansion rates of HA-1- and HA-2-specific CTLs generated byDC. The results indicate that sufficient numbers of CTLs for adoptiveimmunotherapy can be obtained if T-cell cultures will be started with5×10⁷ responder cells. For instance, two HA-2-specific CTLs induced byPBDC showed expansion rates of above nine-, 25- and eight-fold at thesecond, third, and fourth week, respectively. These expansion ratestranslate into an estimated total yield of 3×10⁹−10¹⁰ CTLs at the end ofthe fourth week. The expansion kinetics of the HA-1-specific CTLs wereslower, but the cells expanded consistently with doubling times of twoto three days during each restimulation. It is estimated that 10⁹HA-1-specific CTLs can be obtained after five weeks of culture.

In conclusion, our results show for the first time that mHag HA-1- andHA-2-specific CTLs can reproducibly be generated ex-vivo fromHLA-A2-positive, mHag HA-1- and/or HA-2-negative healthy blood donorsusing dendritic cells pulsed with synthetic peptides. After thesuccessful application of EBV-specific CTLs as specific adoptiveimmunotherapy of EBV-related malignancies,⁴² our results now provide anew possibility for the treatment of relapsed, HA-1- and/orHA-2-positive leukemia patients with HA-1- or HA-2-specific CTLs inducedex-vivo from their HLA identical, mHag-negative bone marrow donors.

Methods

Cell culture: The CD8+ HLA-A2.1 restricted HA-1-specific cytotoxicT-cell clones 3HA15, clone 15 and 5W38 were derived from PBMC of twopatients who had undergone HLA identical bone marrowtransplantation.^(7, 23) The clones were cultured by weekly stimulationwith irradiated allogeneic PBMC and BLCL in RPMI-1640 medium containing15% human serum, 3 mM 1-glutamine, 1% Leucoagglutinin-A and 20 U/mlrIL-2. The HLA-A2.1-positive HA-1 expressing EBV transformed B-celllines (BLCL) Rp and Blk were maintained in IMDM containing 5% FCS. TheKG-1 and T2 cell lines were cultured in 1640 medium containing 3 mM1-glutamine and 10% FCS.

⁵¹Cr-release assay: HPLC fractions and synthetic peptides were tested ina ⁵Cr-release assay as described.²⁴ 2500 ⁵¹Cr labeled T2 cells in 25 mlwere incubated with 25 ml peptide dissolved in Hanks 50 mM Hepes for 30min. at 37° C. Cytotoxic T-cells were added in an end volume of 150 ml.When HPLC peptide fractions were tested, T2 was incubated with 2 mg/mlMA2.1 during the ⁵¹Cr labeling. After 4 hours at 37° C., thesupernatants were harvested.

Peptide purification: Peptides were eluted out of purified HLA-A2.1molecules as earlier described.²⁴ Briefly, HLA-A2.1 molecules werepurified two times from 90×10⁹ HLA-A2.1-positive EBV-BLCL by affinitychromatography with BB7.2 coupled CNBR-activated sepharose 4B beads(Pharmacia LKB) and extensively washed. Peptides were eluted from theHLA-A2.1 with treatment with 10% acetic acid, further acidified by 1%TFA and separated from the HLA-A2.1 heavy chain and b2-microglobulin byfiltration over a 10 kD Centricon (Amicon) filter. Peptides werefractionated using reverse phase micro HPLC (Smart System, Pharmacia).For the first purification, three rounds of HPLC fractionation were usedto purify the HLA-A2.1 restricted HA-1 active peptide fractions from90×10⁹ Rp cells. The first fractionation consisted of buffer A: 0.1%HFBA in H₂O; buffer B: 0.1% HFBA in acetonitrile. The gradient was 100%buffer A (0 to 20 minutes), 0 to 15% buffer B (20 to 25 minutes) and 15to 70% buffer B (25 to 80 minutes) at a flow rate of 100 ml/minute.Fractions of 100 ml were collected. Fraction 24 of the first gradientwas further fractionated. The second fractionation consisted of bufferA: 0.1% TFA in H₂O; buffer B: 0.1% TFA in acetonitrile. The gradient was100% buffer A (0 to 20 minutes), 0 to 12% buffer B (20 to 25 minutes),and 12 to 50% buffer B (25 to 80 minutes) at a flow rate of 100ml/minute. Fractions of 100 ml were collected. A shallower thirdgradient was used to further purify fraction 27 that contained HA-1activity. The gradient was 100% buffer A (0 to 29 minutes), 0 to 18%buffer B (29 to 34 minutes), 18% buffer B (34 to 39 minutes), 18 to23.9% buffer B (39 to 98 minutes) at a flow rate of 100 ml/minute. 1/180to 1/45 of the starting material was used to test for positive fractionsin the ⁵¹Cr-release assay. Comparable HPLC fractionations were used forthe second purification of HLA-A2.1 restricted HA-1 active peptidefractions from 90×10⁹ Blk. 40% of the HA-1 containing fraction 33 of thesecond HA-1 purification was used for an additional reverse phasemicrocapillary HPLC fractionation. Buffer A was 0.1% triethyl amine(TEA) in water buffered to pH 6.0 with acetic acid and buffer B was0.085% TEA in 60% acetonitrile buffered to pH 6.0 with acetic acid. Thegradient was 100% buffer A (0 to 5 minutes), 0 to 100% B (5 to 45minutes) at a flow rate of 0.5 ml/minute. Fractions were collected in 50ml of 0.1% acetic acid every minute for 5 to 15 minutes, every 30seconds from 15 to 20 minutes, every 20 seconds from 20 to 40 minutes,and every 30 seconds from 40 to 45 minutes. For each fraction collected,20% was used to test for HA-1 activity and 80% was used to obtain massspectral data.

Mass spectrometry: Fractions from third dimension HPLC separation of theRp purification that contained the HA-1 activity were analyzed bymicrocapillary HPLC-electrospray ionization mass spectrometry.²⁵Peptides were loaded onto a C18 microcapillary column (75 mm i.d. ×10cm) and eluted with a 34 minute gradient of 0 to 60% B, where solvent Awas 0.1M HOAc(aq) and solvent B was acetonitrile at a flow-rate of 0.5ml/minute. 20% of the effluent was deposited into the wells of a 96-wellplate having 100 ml of culture media in each well (10 second fractions),while the remaining 80% was directed into the electrospray source of theTSQ-70U. Mass spectra and CAD mass spectra were recorded on aFinnegan-MAT TSQ-7000 (San Jose, Calif.) triple quadruple massspectrometer equipped with an electrospray ion source.

HLA-A2.1 peptide binding assay: A quantitative assay for HLA-A2.1binding peptides based on the inhibition of binding of the fluorescentlabeled standard peptide Hbc 18 to 27° F. to C6 (FLPSDCFPSV) (SEQ IDNO:9) to recombinant HLA-A2.1 protein and b2-microglobulin wasused.^(26, 27) In short, HLA-A2.1 concentrations yielding approximately40 to 60% bound fluorescent standard peptide were used with 15 pmol/well(150 nM) b2-microglobulin (Sigma). Various doses of the test peptideswere coincubated with 100 fmol/well (1 nM) fluorescent standard peptide,HLA-A2.1 and b2-microglobulin for one day at room temperature in thedark in a volume of 100 ml in assay buffer. The percent of MHC-boundfluorescence was determined by gel filtration and the 50% inhibitorydose was deduced for each peptide using one-site competition non-linearregression analysis with the prism graph software. Synthetic peptideswere manufactured on an Abimed 422 multiple peptide synthesizer (Abimed,Langenfeld, Del.) and were more than 90% pure as checked by reversephase HPLC.

RT-PCR amplification and sequencing of KIAA0223 region coding for HA-1.:Total or mRNA was prepared from BLCL using the RNAzol method(Cinaa/Biotecx Labs, Houston, Tex.) or according to the manufacturer'sinstructions (QuickPrep mRNA purification Kit, Pharmacia Biotech). cDNAwas synthesized with 1 mg RNA as template and with KIAA0223 basedreverse primer 5′-GCTCCTGCATGACGCTCTGTCTGCA-3′ (SEQ ID NO:16). Toamplify the HA-1 region of KIAA0223, the following primers were used:Forward primer 5′-GACGTCGTCGAGGACATCTCCCAT-3′ (SEQ ID NO:17) and reverseprimer 5′-GAAGGCCACAGCAATCGTCTCCAGG-3′ (SEQ ID NO:18). Cycle parametersused were denaturation 95° C., 1 minute, annealing 58° C., 1 minute andextension 72° C., one minute (25 cycles). The PCR-products were purifiedusing the Magic PCR-Preps DNA purification System (Promega) and directcloned using the pMosBlue T-vector kit (Amersham Life Science). Sixindependent colonies from each individual were sequenced using theT7-sequencing kit (Pharmacia Biotech).

HA-1 allele-specific PCR amplification: In the case of HA-1allele-specific PCR amplification, cDNA was synthesized as describedabove. A PCR amplification was performed with allele-specific forwardprimers: for the HA-1^(H) allele primer H1:5′-CCT-TGA-GAA-ACT-TAA-GGA-GTG-TGT-GCT-GCA-3′ (SEQ ID NO:19), for theHA-1^(R) allele primer R1: 5′-CCT-TGA-GAA-ACT-TAA-GGA-GTG-TGT-GTT-GCG-3′(SEQ ID NO:20) and for both reactions, the reverse primer as describedabove was used. Cycle parameters used were denaturation 95° C., oneminute, annealing 67° C., one minute and extension 72° C., one minute(25 cycles).

Cloning and expression of HA-1^(H) and HA-1^(R) allelic region ofKIAA0223: A forward KIAA00223 based PCR primer containing an ATG startcodon (5′-CCG-GCA-TGG-ACG-TCG-TCG-AGG-ACA-TCT-CCC-ATC-3′ (SEQ ID NO:21))and a reverse KIAA0223 based PCR primer containing a translational stopsignal (5′-CTA-CTT-CAG-GCC-ACA-GCA-ATC-GTC-TCC-AGG-3′ (SEQ ID NO:22))were designed and used in a RT-PCR reaction with cDNA derived from anhomozygous HA-1^(H) and a homozygous HA-1^(R) BLCL. Cycle parametersused were denaturation 95° C., one minute, annealing 60° C., one minuteand extension 72° C., one minute (25 cycles). The desired PCR-productswere purified using the Magic PCR-Preps DNA purification System(Promega). The purified DNA was direct cloned using the pMosBlueT-vector kit (Amersham LIFE SCIENCE) and recloned in the eukaryoticpCDNA3.1(+) vector under the control of a CMV promoter. Transientcotransfections were performed with HLA-A2.1 in Hela cells usingDEAE-Dextran coprecipitation. After three days of culture, HA-1-specificT-cells were added and after 24 hours, the TNFa release was measured inthe supernatant using WEHI cells.²⁸

Peptides: HA-1 and HA-2 peptides were synthesized using a semiautomaticmultiple peptide synthesizer.^(4, 39) The purity of the peptides waschecked by reversed phase high pressure liquid chromatography (HPLC).

APCs: PBMC were isolated by ficoll-hypaque density gradient separationof blood collected with manual hemapheresis.

Monocytes were isolated by plastic adherence during PBDC enrichment.

PBDC were enriched from PBMC by depletion of T-cells, monocytes, B- andNK-cells as described earlier. Briefly, T-cells were depleted by sheepred blood erythrocyte rosetting. Non-T-cells were cultured 36 hours at37° C. in RPMI+10% autologous plasma. After depleting monocytes,non-adherent cells were layered on 14.5% metrizamide gradients andcentrifuged. The light density PBDC were recovered from the interphase.PBDC were identified by FACS being negative for CD3, CD14, CD16 and CD19and positive for HLA-DR. The preparations contained two 6×10⁶ cells witha DC content of 20 to 50%. In some cases the light density cells werefurther depleted from CD14 and CD19 cells using antibody coated magneticbeads.

BMDC were differentiated from bone marrow CD34+ cells (isolated usingCD34+ isolation kit, MACS, Bergisch Gladbach, DE) by culturing with 100ng/ml FLT3-ligand (Genzyme, Leuven; BE), 30 ng/ml IL-3, 25 ng/ml SCF(Genzyme) 50 U/ml TNF-α (Genzyme), 250 U/ml GM-CSF (Genzyme) for ten tofourteen days. The cultures contained 20 to 60% DC as detected by highlevels of DR and negative expression of CD3/CD14/CD16/CD19.

Ex vivo induction of HA-1- and HA-2-specific CTLs: APC were pulsed withHA-1 or HA-2 peptides (both 10 mg/ml) for 90 minutes at 37° C. inserum-free AIM-V medium. After washing, APC and ten 15×10⁶ respondercells (CD4 depleted autologous PBMC) were cultured at different APC:responder cell ratios depending on the type of APC (5:1, 1:3 and 1:10for PBMC, Mo and DC, respectively) in 24-well culture plates. Culturemedium was RPMI supplemented with 10% autologous plasma, 1 U/ml IL-2(Cetus), 1 U/ml IL-12 (Genzyme). The cells were kept at 37° C. in ahumidified, 5% CO₂ air mixture. At day 5, 10 U/ml of IL-2 was added.Starting from day seven, the T-cell cultures were restimulated weeklywith peptide pulsed autologous monocytes. Ten U/ml of IL-2 was added 24hours after each restimulation. The T-cell lines were expanded with 10to 20 U/ml IL-2 containing culture medium.

Cytotoxicity (⁵¹Cr release) assays: Standard 4-hour ⁵¹Cr release assaysusing PHA-blasts, EBV-BLCL and fibroblasts and leukemic cells as targetcells were performed as described before.⁸ The percent specific lysiswas calculated using the following formula: 100×(cpm experimentalrelease-cpm spontaneous release)/(cpm maximal release-cpm spontaneousrelease).

Target cells: EBV-BLCL were generated as described before⁸ and culturedin RPMI plus 10% FCS. PHA-activated T-cell blasts (PHA-blasts) wereobtained by stimulation of PBMC with 0.1 mg/ml PHA (Wellcome) for 72hours. PHA-blasts were expanded with medium containing 20 U/ml IL-2.Skin fibroblasts of an HLA-A2+, HA-1+, HA-2+ healthy individual wereisolated, cultured and tested as described before.⁸ Fibroblasts weretrypsinized and cultured in the wells of 96-well flat-bottomedmicrotiter culture plates at a concentration of 3×10³ cells/well with orwithout addition of IFN-γ and TNF-α (both 300 U/ml) for 72 hrs. Whenindicated, target cells were pulsed with HA-1 or HA-2 peptides (both 10mg/ml) during ⁵¹Cr labeling.

Leukemia patients' (AML or ALL) PBMC or BM containing >95%morphologically recognizable malignant cells were assigned as leukemiccells. Leukemic cells were thawed and cultured in RPMI plus 10% humanserum for 72 hours with or without addition of IFN-γ and TNF-α (both 300u/ml) before using as target cells.

In vivo induced mHag-specific T-cell clones: In vivo induced, mHag HA-1-and HA-2-specific CD8+ CTL clones were isolated from post BMT leukemiapatients, and were documented in detail.³⁵

Example 2

To confirm the hematopoietic system restricted tissue distribution,earlier analyzed by HA-1-specific CTLs, HA-1 mRNA levels were analyzedby quantitative real-time PCR (Example 4) in eight differenthematopoietic and six different non-hematopoietic cell types. Only cellsof hematopoietic origin expressed significant levels of the HA-1 gene(FIG. 8). No significant HA-1 gene expression was detected in cells ofnon-hematopoietic origin: i.e., keratinocytes, dermal fibroblasts,proximal tubular epithelial cells (PTECs), human umbilical veinendothelial cells (HUVECs), melanocytes and SV 40 immortalized breastcell lines HaCaT and HBL 100 (FIG. 8).

Next, we investigated the HA-1 gene transcription levels in 35epithelial tumor cell lines derived from different carcinomas (Table 1).The HA-1 gene transcription, analyzed by quantitative real time RT-PCR,revealed significant HA-1 mRNA in 26 out of the 35 cell lines of variousmalignant origins. Table 1 also lists the results of the commonleukocyte antigen CD45. We compared the HA-1 and CD45 RNA expression invarious hematopoietic cells. Both genes are expressed in hematopoieticcells to comparable levels (data not shown). None of the tumor celllines showed significant CD45 gene expression. This shows that HA-1transcription observed in the tumor cell lines is specific and not dueto contaminating HA-1-positive hematopoietic cells (Table 1).

Functional recognition by HA-1-specific CTLs is a prerequisite fortumor-specific targeting in immunotherapeutic settings. The mHag HA-1locus encodes two alleles, i.e., the HA-1H and the HA-1R allele. TheHA-1H allele is the T-cell epitope that is recognized by the HLA-A2restricted CTL.⁵² Therefore, we executed CTL recognition studies(Example 5) on the tumor cell lines that expressed both the HLA-A2restriction molecule and the HA-1H T-cell epitope required for theHLA-A2 restricted HA-1-specific CTL recognition. Hereto, all tumor celllines listed in Table 1 were HLA and HA-1 genotyped.⁵² Table 2 showssignificant HA-1 CTL lysis on four of the five cell lines by twoHA-1-specific clones which could be enhanced in all cases by IFNγ andTNFα treatment of the target cells. The colon carcinoma cell line CaCo-2was only recognized by one of the two HA-1 CTL clones.

With the demonstrated functional expression of HA-1 by epithelial tumorcell lines, we expected that HA-1 is also expressed by epithelial tumorsin vivo. However, given the expression of HA-1 by cells of thehematopoietic lineage and in view of the virtual omnipresence ofhematopoetic cells in tumors, spurious positive results of a PCRanalysis caused by contaminating hematopoietic cells should be avoided.To this end, we applied laser-mediated micro-dissection to cryosectionsof fresh frozen cancer samples without any microscopically visibleleukocyte infiltration (Example 6, FIG. 9A). As control, we usedmicro-dissected normal breast glands from three patients that underwentbreast reduction surgery (FIG. 9B). By the applied micro-dissectionmethod the selected area is cut by a laser beam and directly catapultedinto the reaction tube, practically excluding contamination bysurrounding tissue. Of twelve tumors obtained from patients with breastand lung cancers and the three biopsies from normal breast tissue, areasof 10,000 to 60,000 μm² in total (comprising about 30 to 200 cells) werelaser-micro-dissected. mRNA was isolated, reverse transcribed andamplified with a recently developed global amplification method (Example7). Successful global amplification of cDNA was checked by establishedgene-specific amplification of the two housekeeping genes b-actin andEF-1a and cDNA array hybridization (not shown). Following dilution ofthe primary PCR products, specific primers served to detect HA-1 geneexpression (Example 8). While 7 of twelve tumors were positive for HA-1,all normal breast glands were negative (FIG. 9C). The identity of thePCR bands as HA-1 was confirmed by Southern blotting (not shown). Weused CD45 gene-specific PCR to test whether HA-1 expression might beattributed to single infiltrating leukocytes or intravascular cells thathad escaped our attention. Absence of CD45 mRNA would provide strongevidence that the HA-1 signal originates from the epithelial tumor cellsin vivo. Indeed, four of 7 tumor samples solely expressed HA-1 (FIG. 9C,arrows) in at least one of the micro-dissected areas, whereas threetumors co-expressed CD45 and HA-1 prohibiting evaluation of their HA-1status. Therefore, HA-1 was found to be expressed in at least 30% humanprimary tumors of epithelial origin in vivo.

Since contamination by CD45-positive non-epithelial cells could not beabsolutely excluded as cause of the encountered CD45 expression in someof the micro-dissected tumor areas, we resorted to HA-1 analysis ofsingle tumor cells or defined cell clusters freshly isolated from bonemarrow or lymph nodes of cancer patients (FIG. 10A). Single disseminatedcancer cells were detected in cell suspensions prepared from bone marrowand lymph node samples with a fluorescent labeled monoclonal antibodyagainst the epithelial cell adhesion molecule (EpCAM) as marker.⁵³ Intotal, twenty-seven single tumor cells or small cell clusters wereisolated by micromanipulation from 15 cancer patients (FIG. 10A). ForcDNA analysis, the same global amplification technique was applied thatwas used for the micro-dissected tumor areas, enabling faithfuldetection of expressed transcripts in single cells (Example 7). Thelabeled cDNAs were hybridized to an array including specific epithelialmarker genes such as the cytokeratin family members (KRT), mammaglobin(MBG) and prolactin induced protein (PIP) as markers for breast-derivedcells, and the transcription factor ELF3. Further evidence of epithelialorigin was provided by claudin 7 (CLDN7) and desmoplakin I (DSP) bothinvolved in epithelial cell adhesion. As indicator of malignancy, theexpression of MAGE genes was analyzed, the transcripts are found inspermatogonal cells and exclusively in various cancer cells, hence thedesignation cancer-testis genes. In addition, we evaluated the cells formarkers of hematopoietic cells such as the T-cell receptor, CD45, CD33,CD34, CD37, CD38, and CD16. The isolated cells expressed none of thehematopoietic markers (not shown). Expression of cytokeratins and otherepithelial markers indicated their epithelial origin (FIG. 10B). In somecases the cells were positive for one or more MAGE genes suggestingtheir tumor origin, despite down-regulation of cytokeratin mRNA (FIG.10B). All cells were then tested for HA-1 and CD45 expression bygene-specific PCR; the HA-1 amplification products were subsequentlyconfirmed by restriction enzyme digest and by Southern blotting (Example8). Six of the 27 cells expressed the HA-1 gene and none of themexpressed the CD45 gene (FIG. 11). The HA-1 significant transcripts wereobserved in samples derived from breast cancer (PN4-C1), bronchialcarcinoma (PN3-C1, PN5-C1, PN6-C5), prostate cancer (PN2-C1) andcervical cancer (PN1-CI). From two of the HA-1-positive cells (PN5-C4,PN3-C1) we could besides mRNA also evaluate their DNA by a recentlydescribed method.⁵³ The isolated DNA was subjected to whole genomeamplification and comparative genomic hybridization (CGH). Both cellsharbored multiple genomic alterations, lending ultimate proof of theirmalignant nature (FIG. 12).

Example 3

We have investigated whether the HA-1^(H/R) polymorphic region containspeptides that can be presented by other HLA molecules than HLA-A2.Hereto, we analyzed the binding capacities of HA-1 polymorphic peptidesto nine HLA-A and -B molecules that have a frequency of more than 10% inthe Caucasian population. Nonameric HA-1^(H/R) peptides (n=18) weretested for binding to these frequent HLA alleles. The peptide bindinganalyses were extended with two decameric HA-1^(H/R) peptides thatcontained binding motives for HLA-A3 and with five nonameric/decamericpeptides that were predicted to bind to HLA-B14 or to -B60. Next to thebinding studies, cellular processing was executed by in vitro proteasomedigestion of 29 amino acid long HA-1^(H) and HA-1^(R) peptides. Toenlarge the patient population for HA-1-specific immunotherapy, theHLA-B60 binding peptides were analyzed for their in vitro immunizingpotential. Hereto, peptide loaded dendritic cells (DCs) were used toinduce T-cell responses from healthy individuals.

Material and Methods

HA-1 peptides: HA-1^(H) and HA-1^(R) peptides were synthesized using anautomated multiple peptide synthesizer (Syro II, Multisyntech, Witten,Del.) according to the known HA-1 amino acid sequence.⁶¹ The purity ofthe peptides was >90%. The peptides were dissolved in dimethyl sulfoxide(DMSO), diluted in 0.9% NaCl and stored at −20° C. until use.

Prediction of HLA peptide binding: The polymorphic HA-1^(H) and HA-1^(R)regions were screened with the HLA-peptide binding prediction softwareof BIMAS (BioInformatics & Molecular Analysis Section, NIH, Bethesda,Md.; url: bimas.dcrt.nih.gov for octameric, nonameric or decameric HA-1peptides capable to bind to HLA class-I molecules. The selection ofpeptide candidates was made by comparison of the computed scores withthat of the HLA-A2 restricted HA-1^(H) CTL epitope with amino acid (aa)sequence VLHDDLLEA (SEQ ID NO:2) (score=79.6). This score corresponds tothe estimated half-time of dissociation of complexes containing thepeptide at 37° C. at pH 6.5. Five HA-1^(H/R) peptides with scoresranging from 32 (intermediate binding score) to 176 (strong bindingscore) were selected to assay for binding to the relevant HLA class-Imolecules. The predicted HLA class-I /HA-1^(H/R) peptide associationsand their computed binding scores are presented in Table 3. In addition,we selected two decameric HA-1^(H/R) peptides that contained anchorresidues for binding to HLA-A3 but were not predicted by the BIMASsoftware. With reference to Table 3, peptide number 1 is represented bySEQ ID NO:34, peptide number 2 is represented by SEQ ID NO:35, peptidenumber 3 is represented by SEQ ID NO:36, peptide number 4 is representedby SEQ ID NO:37, peptide number 5 is represented by SEQ ID NO:38,peptide number 6 is represented by SEQ ID NO:39, peptide number 7 isrepresented by SEQ ID NO:4, peptide number 8 is represented by SEQ IDNO:5, peptide number 9 is represented by SEQ ID NO:6, peptide number 10is represented by SEQ ID NO:7, peptide number 11 is represented by SEQID NO:40, peptide number 12 is represented by SEQ ID NO:23, peptidenumber 13 is represented by SEQ ID NO:41, peptide number 14 isrepresented by SEQ ID NO:42, peptide number 15 is represented by SEQ IDNO:2, peptide number 16 is represented by SEQ ID NO:10, peptide number17 is represented by SEQ ID NO:43, peptide number 18 is represented bySEQ ID NO:44, peptide number 19 is represented by SEQ ID NO:45, peptidenumber 20 is represented by SEQ ID NO:46, peptide number 21 isrepresented by SEQ ID NO:47, and peptide number 22 is represented by SEQID NO:48.

HLA peptide binding assays: We used the competition-based HLA peptidebinding assay as described previously, with some modifications.⁶⁷Briefly, HLA typed EBV-LCLs were washed with PBS, kept on ice for fiveminutes and treated with an ice-cold 0.132 M citric acid, 0.062 MNa₂HPO₄.2H₂O elution buffer for 90 seconds.⁶⁷ The pH of the elutionbuffer was optimized for each HLA molecule to enable maximal elution ofHLA bound peptides (manuscript in preparation). Immediately after mildacidic treatment, the cells were washed with 12 ml Iscove's modifiedDulbecco's medium (IMDM, Bio Whittaker, Belgium) containing 2% FCS andresuspended in IMDM containing 2% FCS, 1.5 μg/ml β2 microglobulin(Sigma, St. Louis, Mo., US). 4×10⁴ acid treated EBV-LCLs were thenincubated in 96-well V-bottom plates (Costar, Cambridge, Mass., US) withfluorescent-labeled reference peptide (25 μl/well, final concentration:150 nM) mixed with serial dilutions of competitor (test) peptides (25μl/well; final concentrations: 100 to 0.78 μM) in a total volume of 150ml. All reference peptides were deduced from previously reportedpeptides that show strong binding to the respective HLA class-Imolecules.⁶⁸ After incubation for 24 hours at 4° C., the cells werewashed twice with 100 μl/well PBS/1% FCS and fixed with 0.5%paraformaldehyde in PBS. The mean fluorescence expressed by the cellswas determined by a FACScalibur flow cytometer (Becton-Dickinson, St.Louis, Mo., US). Percentage inhibition of the HLA binding of thefluorescent reference peptide is calculated with the formula: %inhibition=1−[(mean fluorescence in the presence of competitorpeptide−mean background fluorescence)/(mean fluorescence in the absenceof competitor peptide−mean background fluorescence.)]×100%. The relativebinding affinity of the peptides is expressed as the peptideconcentration that inhibits 50% of the binding of the reference peptide(IC₅₀).

Proteasomal cleavage of the HA-1 polymorphic region: Twenty-nine aminoacid long HA-1^(H) and HA-1^(R) peptides were purified to >95% byreverse phase HPLC. 10 μg/ml of the peptides were incubated with 20Sproteasomes isolated from EBV-LCLs for 15 minutes, 30 minutes; 45minutes as described elsewhere.⁶⁹⁻⁷¹ The proteolysis products wereanalyzed by tandem mass spectrometry, as described.⁷²

Dendritic cell culture: Monocyte-derived DCs (MoDCs) were generated fromhealthy individuals by culturing peripheral blood-derived CD14+monocytes with 1000 U/ml IL-4 (Genzyme, Cambridge, Mass., US) and 800U/ml GMCSF (donated by Dr. S. Osanto, LUMC, Leiden, NL) for six days asdescribed elsewhere.⁷³ On day 6, the DCs were maturated by culturing onirradiated (750 gy) CD40L transfected mouse fibroblasts at a DC tofibroblast ratio of 2:1 or by adding 50% of monocyte conditionedmedium.⁷³ Mature DCs were pulsed with HA-1 peptides for two hours at 37°C. in Aim-V medium prior to their use as stimulator cells.

In vitro induction of HLA-B60/HA-1-specific T-cell responses: Peptidepulsed DCs were cocultured with autologous PBMC at a DC to PBMC ratio of1:10 in IMDM, 10% human serum supplemented with 1 U/ml IL-2 (Cetus,Emeryville, Calif., US) and 1 U/ml IL-12 (R & D systems, Minneapolis,Minn., US). On day 5, 20 U/ml IL-2 was added. On day 7, the T-cell lines(TCL) were depleted of CD4+ cells using immunomagnetic beads (Dynal AS,Oslo, Norway) and were restimulated with irradiated (150 Gy) peptidepulsed mature DCs (DC:T-cell ratio 1:10) or with irradiated (150 Gy)peptide pulsed monocytes (monocyte:T ratio=1:3). Twenty-four hours and96 hours after restimulation, medium containing 20 U/ml IL-2 was added.TCL were subsequently restimulated every 7 days and were tested forHA-1-specific activity in Interferon-γ (IFN-γ) elispot assays⁷⁴ prior toeach restimulation.

RESULTS: Effective binding of nonameric and decameric HA-1^(H) andHA-1^(R) peptides to HLA-B60: Three categories of HLA molecules wereselected for the peptide binding assays: those molecules with afrequency of more than 10% in the Caucasian population, those withbinding motifs and those that were predicted to bind nonameric/decamericHA-1^(H/R) peptides. All nonameric HA-1^(H) and HA-1^(R) peptides (n=18)were tested for binding to the so called frequent HLA class-I moleculesHLA-A1, -A2, -A3, -A11 , -A24, -B7, -B8, -B35, -B62. The peptideanalysis was extended with two decameric HA-1^(H/R) peptides with abinding motif for HLA-A3 and with five nonameric/decameric peptidespredicted to bind either to HLA-B14 or -B60 (Table 3). The HLA-A1, -A11,-A24, -B7, -B8, -B14, -B35 and -B62 molecules did not bind nonamericHA-1^(H/R) peptides, despite the predictions of BIMAS software forintermediate to strong binding of peptide ECVLRDDLL (SEQ ID NO:23) toHLA-B8 or to -B14 (Table 3). The decameric HA-1^(H/R) peptidesVLH/RDDLLEAR showed weak to intermediate binding to HLA-A3 moleculeswith IC₅₀ values of 15.6 μM and 37.5 μM respectively (FIG. 13). Inagreement with the prediction of the BIMAS software, the nonameric anddecameric HA-1^(H/R) peptides KECVLHDDL (SEQ ID NO:4), KECVLRDDL (SEQ IDNO:5), KECVLHDDLL (SEQ ID NO:6) and KECVLRDDLL (SEQ ID NO:7) showedstrong binding to HLA-B60 molecules with very low IC₅₀ values of 5.3 μM,3.9 μM, 1.0 μM and 1.6 μM respectively (FIG. 14). As expected, theoriginal HLA-A2/HA-1^(H) CTL epitope, also predicted by the BIMASsoftware, displayed binding to HLA-A2 with an IC₅₀ value of 6.4 μM (datanot shown).

Stable binding of nonameric and decameric HA-1^(H) and HA-1^(R) peptidesto HLA-B60: The stability of the HLA-B60/HA-1^(H/R) peptide binding wasaddressed by testing for the HLA peptide binding capacities at 4° C. and25° C. HLA-A2/HA-1^(H/R) peptide binding stability was analyzed inparallel as comparison. Increasing the temperature from 4° C. to 25° C.did not affect the strong binding of decameric HA-1^(H/R) peptides toHLA-B60 (FIG. 15A). Less binding was observed with the nonamericHA-1^(H/R) peptides to HLA-B60 (FIG. 15B) which was comparable to thenonameric HA-1^(H) peptide to HLA-A2 (FIG. 15C). Increasing thetemperature from 4° C. to 25° C. further decreased the intermediatebinding of the nonameric HA-1^(R) peptide to HLA-A2 (FIG. 15C). Thus,the binding of both HA-1^(H) and HA-1^(R) peptides to HLA-B60 werestable and not temperature sensitive.

Proper proteasomal cleavage of the HLA-B60 binding HA-1^(H/R) peptides:Twenty-nine amino acid long HA-1^(H/R) peptides were subjected to invitro digestion with EBV-LCL-derived 20S immuno-proteasomes. Within atime frame of 15 minutes, major peptide fragments were cleaved at theCOOH-termini of both nonameric and decameric HLA-B60 binding HA-1^(H/R)peptides. The latter cleavage products contained the intact HLA-B60binding sequences with three to five additional amino acid residues atthe N termini for the HA-1^(H) and HA-1^(R) peptides as demonstrated inTable 4 and Table 5, respectively. Thus, both the HA-1^(H) and theHA-1^(R) products can be effectively cleaved by proteasomes to generatethe precursors of the peptides that bind to HLA-B60. A 29 amino acidlong HA-1^(A) peptide is represented by SEQ ID NO:78. Fragments 1through 9 in Table 4 are represented by SEQ ID NOS:79-87, respectively.A 29 amino acid long HA-1^(R) peptide is represented by SEQ ID NO:88.Fragments 1 through 13 of Table 5 are represented by SEQ ID NOS:89-101.

In vitro induction of HLA-B60 restricted T-cells against the nonamericHA-1^(H) peptide: To test the immunogenicity of both the HA-1^(H) andthe HA-1^(R) peptides in the context of HLA-B60, PBMCs from threeHLA-B60⁺ HA-1^(RR) and from two HLA-B60⁺ HA-1^(HH) healthy individualswere stimulated with autologous DCs pulsed with the nonameric HA-1^(H)or HA-1^(R) peptide, respectively. After two or three rounds ofstimulation, the two T-cell lines (TCL) induced with the HA-1^(R)peptide contained significant numbers of IFN-γ producing T-cells thatrecognized HA-1^(R) peptide pulsed HLA-B60 transfected T2 cells.Nevertheless, neither TCL induced with HA-1^(R) peptide produced IFN-γupon stimulation with EBV-LCLs that express the natural ligandHLA-B60/HA-1^(R) (data not shown). On the contrary, all three TCLinduced with the HA-1^(H) peptide contained, aside from HA-1non-specific T-cells, a significant number of T-cells that producedIFN-γ not only upon stimulation with HA-1^(H) peptide pulsed HLA-B60transfected T2 cells, but also upon stimulation with EBV-LCLs thatexpress the natural HLA-B60/HA-1^(H) ligand (FIG. 16).

In search for novel T-cell epitopes in the HA-1^(H/R) polymorphicregion, we studied the binding of polymorphic HA-1 peptides to 11 HLAclass-I molecules and analyzed the proteasomal cleavage sites in theHA-1^(H/R) polypeptides. These analyses suggested novel interactions ofboth alleles of the mHag HA-1 locus with HLA-B60 molecules. Bothnonameric and decameric HA-1^(H/R) peptides effectively bind to HLA-B60.In vitro proteasomal analysis showed cleavage at the COOH termini ofHLA-B60 binding peptides, indicating proper intracellular processing.

Both nonameric and decameric HA-1^(H/R) peptides show strong binding toHLA-B60, with IC₅₀ values between 1.6 to 5.3 μM. These HLA bindinglevels are similar to or higher than the HLA binding of the immunogenicHLA-A2/HA-1^(H) CTL epitope and of other reported T-cell epitopesmeasured in similar assays.^(67, 75) Furthermore, we compared thestability of the HLA-B60/HA-1^(H/R) with HLA-A2/HA-1^(H/R) peptideinteractions by increasing the temperature of the binding assays. Theseassays reveal that unlike the HLA-A2/HA-1^(R) peptide interaction, theHLA-B60/HA-1^(H/R) and HLA-A2/HA-1^(H) interactions are stable. Thestability of HLA-B60/HA-1^(H/R) interactions were confirmed in separateexperiments using fluorescent HA-1^(H/R) peptides (data not shown).Thus, both HA-1^(H) and HA-1^(R) peptides can efficiently interact withHLA-B60, which is an important biochemical feature of stronglyimmunogenic T-cell epitopes.⁷⁵ This actually predicts immunogenicity ofboth HA-1^(H) and HA-1^(R) locus products in association with HLA-B60.

The HLA peptide binding is preceded by intracellular processing ofcellular proteins. In the endoplasmic reticulum (ER), proteasomallycleaved peptides can undergo NH2-terminal trimming by aminopeptidases.⁷⁶COOH-terminal trimming in the ER have not been demonstrated. The propergeneration of the correct COOH terminus by an early major cleavage siteby proteasomes is thus a key event for efficient epitope generation asdemonstrated by recent studies.⁷⁷⁻⁸⁸ In our in vitro cleavage studies,the correct COOH termini of HLA-B60 binding sequences of both theHA-1^(H) and the HA-1^(R) allele were generated within 15 minutes. Thesepeptide fragments contained the intact HLA-B60 binding sequences. Theexact sequences of the HLA-B60 binding peptides were not present asproteasomal degradation products. Also some additional cleavage siteswithin the putative T-cell epitopes were observed. Nonetheless, thesuccessful generation of HLA-B60/HA-1^(H)-specific T-cells demonstratesthe proper cleavage of the HLA-B60 binding HA-1^(H) peptides by cellularantigen processing machinery.

Example 4

Total RNA was prepared from subconfluent layers of the adherent cellcultures using the RNAzol method (Cinaa/Biotecx Laboratories, Houston,Tex.) according to the manufacturer's description. cDNA was synthesizedusing 2 mg RNA and random hexameric primers. PCR amplification andquantification were performed using the Taqman PCR assay (PE AppliedBiosystems 7700 Sequence Detector, Foster City, Calif.). We usedcomparative quantification normalizing the HA-1 and CD45 gene to aninternal standard gene, the ubiquitously expressed housekeeping geneporphobilinogen deaminase (PBGD). To allow calculation of relativelevels of expression, we used the KG-1 cell line, which expresses bothgenes, as a standard. The HA-1 and CD45 expression levels of the testsamples were calculated as percentages of HA-1 and CD45 expressionlevels in the reference cell line KG-1. All samples tested which showedexpression levels below 10% in the real time quantitative PCR did notproduce detectable PCR fragments in a standard PCR. Therefore,expression levels <10% are considered as not significant. The relativequantification was calculated by the linear calibration function betweenthe threshold cycle (Ct) value and the logarithm of the initial startingquantity (N) were Ct=−3.31 log (N)+26.1, Ct=−3.5 log(N)+21.6 andCt=−3.41 log(N)+25.6 for HA-1, CD45 and PBGD, respectively. The HA-1,CD45 and PBGD expression were quantified in all test samples by usingthese calibration functions.

Example 5

Tumor cell lines were used as target cells in a four-hour ⁵¹Cr releaseassay. The tumor cells from subconfluent cultures were harvested anddispensed at 2500 cells/well in 96-well flat-bottomed microtiter platesand allowed to attach either in the presence or the absence of rINFγ(250 U/ml, Gentech, San Francisco, Calif.) and TNFα (250 U/ml) for 48hours. The tumor cells were labeled with ⁵¹Cr for one hour and theexperiments were performed in sixplicates. The percentage specific lysiswas calculated as follows: % specific lysis=(experimentalrelease/spontaneous release)/(maximal release/spontaneous release)×100.

Example 6

Preparation of cryosections: Sections (5 μm) from freshly shock frozenprimary tumors were placed on a polyethylene membrane on a glass slide,stained with Meyer's hematoxylin and dehydrated in 70%, 90% and 100%ethanol. The PALM Microbeam system (Bemried, Del.) was used formicro-dissection and catapulting.

Example 7

Detection of disseminated cells, global amplification of micro-dissectedareas and of single cells from bone marrow and lymph nodes was performedas described in detail (Klein et al., submitted). Briefly, the viablebone marrow or lymph node samples were stained for ten minutes with 10μg/ml monoclonal antibody 3B10-C9 in the presence of 5% AB-serum.3B10-C9-positive cells were detected with B-phycoerythrin-conjugatedgoat antibody to mouse IgG (The Jackson Laboratory) and transferred toPCR-tubes on ice. Oligo-dT beads in 10 μl lysis buffer (Dynal) wereadded, the cells lysed, tubes rotated for 30 minutes to capture mRNA.Ten μl cDNA wash buffer-1 (50 mM Tris-HCl, pH 8.3, 75 mM KCI, 3 mMMgCl2, 10 mM DTT, supplemented with 0.5% Igepal (Sigma)) was added andmRNA bound to the beads washed in 20 μl cDNA wash buffer-2 (50 mMTris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, supplemented with0.5% Tween-20 (Sigma)), transferred to a fresh tube and washed again incDNA wash buffer-1. mRNA was reverse transcribed with Superscript IIReverse Transcriptase (Gibco BRL) using the buffers supplied by themanufacturer supplemented with 500 μM dNTP, 0.25% Igepal, 30 μM CFL5c8primer (5′-(CCC)5 GTC TAG ANN (N)8-3′ (SEQ ID NO:25)) and 15 μM CFL5cT(5′-(CCC)5 GTC TAG ATT (TTT)4 TVN (SEQ ID NO:26), at 44° C. for 45minutes. Samples were rotated during the reaction to avoid sedimentationof the beads. cDNA remained linked to the paramagnetic beads via themRNA and was washed once in the tailing wash buffer (50 mM KH2PO4, pH7.0, 1 mM DTT, 0.25% Igepal). Beads were resuspended in tailing buffer(10 mM KH2PO4, pH 7.0, 4 mM MgCI2, 0.1 mM DTT, 200 μM GTP) and cDNA-mRNAhybrids were denatured at 94° C. for four minutes, chilled on ice, 10 UTdT (MBI-Fermentas) added and incubated at 37° C. for 30 to 60 minutes.After inactivation of the tailing enzyme (70° C., five minutes), PCR-MixI was added consisting of 4 μl of buffer-1 (Roche, Taq long template),3% deionized formamide (Sigma) in a volume of 35 μl. The probes wereheated at 78° C. in the PCR cycler (Perkin Elmer 2400), PCR Mix II,containing dNTPs at a final concentration of 350 μM, CP2 primer(5′-TCA-GAA-TTC-ATG-CCC-CCC-CCC-CCC-CCC-3′ (SEQ ID NO:27), finalconcentration 1.2 μM) and 5 Units of the DNA Poly-Mix was added, (Roche,Taq Long Template) in a volume of 5 μl for a hot start procedure. 40cycles were run at 94° C. for 15 seconds, at 65° C., 30° C, 68° C. fortwo minutes for the first 20 cycles and a ten-second elongation of theextension time each cycle for the remaining 20 cycles, and a finalextension step at 68° C. for 7 minutes.

For expression profiling, digoxigenin-UTP was incorporated by PCR using0.1 to 1 μl of the original PCR amplified cDNA fragments reamplificationin the presence of 50 μM dig-dUTP (Roche), 300 μM dTTP, and other dNTPsat a final concentration of 350 μM. Reamplification conditions wereessentially as described above, modifications were the use of 2.5 Unitsof the DNA Poly Mix. Initial denaturation at 94° C. for two minutesfollowed by 12 cycles at 94° C., 15 seconds, 68° C., three minutes and afinal extension time of 7 minutes. Filters were pre-hybridized overnightin the presence of 50 mg/ml E. coli and 50 mg/ml pBS DNA in 6 mlDig-easy Hyb buffer (Roche). Labeled PCR products from single cells wereadded in a concentration of 1.5 μg/ml mixed with 100 μg herring sperm toprehybridization buffer, and hybridized for 36 to 48 hours. Stringencywashes were performed according to the Roche™ digoxigenin hybridizationprotocol adding two final stringency washes in 0.1×SSC+0.1% SDS for 15minutes at 68° C. Detection of filter bound probes was performedaccording to the digoxigenin detection system protocol supplied with thekit (Roche).

Example 8

Amplification of HA-1 and CD45. All samples were analyzed by two primerpairs for HA-1: HA-1 (I) (forward: 5′-GAC GTC GTC GAG GAC ATC TCC CAT-3′(SEQ ID NO:17); reverse: 5′-GAA GGC CAC AGC AAT CGT CTC CAG-3′ (SEQ IDNO:18)) and HA-1 (II) (forward: 5′-ACA CTG CTG TCG TGT GAA GTC-3′ (SEQID NO:29); reverse: 5′-TCA GGC CCT GCT GTA CTG CA-3′ (SEQ ID NO:30)).CD45 forward: 5′-CTG AAG GAG ACC ATT GGT GA (SEQ ID NO:31)) and reverse:5′-GGT ACT GGT ACA CAG TTC GA-3′ (SEQ ID NO:32) primer. Amplificationproducts of the HA-1 (I) primers were digested with the restrictionenzyme BstU I and amplification products of the HA-1 (II) primers withHinf I. Southern blot was performed according to standard protocols.

TABLE 1 HA-1 and CD45 gene expression in tumor cell lines. Percentagesrepresent HA-1 and CD45 gene expression relative to the standard cellline KG-1 as analyzed by quantitative real time PCR. % Tumor type Cellline % CD45 HA-1 Breast cancer ZR75-1 ≦10 54 BT-20 ≦10 40 734B ≦10 27T47 D ≦10 17 MDA-MB231 ≦10 15 MCF-7 ≦10 ≦10 BT 474 ≦10 ≦10 Melanoma Mel93.04 ≦10 68 KUL 68/3636 ≦10 67 BB 74/2940 ≦10 57 MNT ≦10 27 LB33 ≦10 24BT ≦10 15 453 Ao ≦10 12 518A ≦10 ≦10 E9 ≦10 ≦10 MEWO ≦10 ≦10 Lungcarcinoma GLC 36 ≦10 22 GLC 8 ≦10 ≦10 GLC 2 ≦10 ≦10 Renal Cell CarcinomaMZ 1851 ≦10 29 MZ 1752 ≦10 13 MZ 1774 ≦10 ≦10 BA ≦10 ≦10 Hepatoma HuH7≦10 37 HepG2 ≦10 35 Colon carcinoma SW 707 ≦10 147 CaCo-2 ≦10 81 SW 480≦10 70 SW 2219 ≦10 48 SW 620 ≦10 28 Col 205 ≦10 21 SW 948 ≦10 12 HT29≦10 11 Head and Neck cancer BB 49/1413 ≦10 54

TABLE 2 CTL recognition of tumor cell lines. The results are given aspercentage specific lysis (Example 5) by one allo HLA-A2 and by two HA-1specific CTL clones at different effector (E) to target (T) ratios. %specific lysis by HLA-A2 HA-1 CTLs CTLs 5W38 3HA15 Tumor cell linesIFNγ/TNAα IFNγ/TNFα IFNγ/TNFα Tumor type Designation E:T no yes no yesno yes Breast cancer MDA-MB 231  2:1 10 13 8 15 8 13 10:1 50 64 31 47 2539 Melanoma MEL 93.04  2:1 10 14 1 13 −2 10 10:1 54 64 12 37 17 40Melanoma 453 A0  2:1 7 24 1 10 1 18 10:1 25 43 5 21 2 22 20:1 35 45 7 247 21 Lung carcinoma GLC 36  1:1 33 35 6 12 0 8 10:1 59 80 8 25 17 25Colon carcinoma CaCo-2 1.6:1  20 22 1 2 6 7 16:1 29 49 4 4 11 17

TABLE 3 Peptides of the HA-1 polymorphic region tested for binding todifferent HLA class I molecules. Pep- ide HA-1^(H/R) polymorphic regionsequence Binding predicted to No. E K L K E C V L H/R D D L L E A R R(BIMAS score)  1 E K L K E C V L H  2 E K L K E C V L R  3   K L K E C VL H   D  4   K L K E C V L R   D  5     L K E C V L H   D D  6     L K EC V L R   D D  7       K E C V L H   D D L HLA-B60 (176)  8       K E CV L R   D D L HLA-B60 (176)  9       K E C V L H   D D L L HLA-B60 (160)10       K E C V L R   D D L L HLA-B60 (160) 11         E C V L H   D DL L 12         E C V L R   D D L L HLA-B8 (32), -B14 (90) 13           CV L H   D D L L E 14           C V L R   D D L L E 15             V LH   D D L L E A HLA-A2 (79.6) 16             V L R   D D L L E A 17            V L H   D D L L E A R HLA-A3 18             V L R   D D L LE A R HLA-A3 19               L H   D D L L E A R 20               LR   D D L L E A R 21                 H   D D L L E A R R 22                R   D D L L E A R R Peptide Nos. 1-8, 11-16 and 19-22were assayed for binding to HLA-A1, -A2, -A3, -A11, -A24, -B7, -B8,-B35, -B62, regardless of prediction. ¹Prediction of HLA/peptideassociations was executed using BIMAS software except for peptides 17and 18, which were not predicted by BIMAS but contain HLA-43 anchoramino acids at position 2 and 10.

TABLE 4 In vitro proteasomal cleavage of a 29 amino acid longHA-1^(A) peptide % fragment digested in 15 min 30 min 45 min G L E K LK E  C V  L H  D  D L  L E A R R P R A H E C L G E A 17.2 22.4 0 G L EK L K E C V L H D D L 14.7 11.8 14.7 G L E K L K E C V L H D D L L E A RR P R A H E C L G 13.9 16.4 21.7                     H D D L L E A R R PR A H E C L G E A 13.0 10.3 13.9 G L E K L K E C V L H D D L L E A R R PR A 10.5 8.3 12.1     E K L K E C V L H D D L L  9.6 8.8 12.3 G L E K LK E C V L H D D L L E A R R P R A H E C  8.5 9.2 13.8 G L E K L K E C VL H D  7.8 7.4 11.5 G L E K L K E C V L H D D L L E A  4.8 5.5 0 G L E KL K E C V L The peptide sequences that bind to HLA-B60 are underlined.The proteolytic fragments cleaved at the COOH termini of the HLA-B60binding peptides are indicated in bold. The amounts of the generatedfragments after cleavage with 20s immuno proteasomes for 15, 30 and 45minutes are expressed as the percentage of all fragments found in thedigested substrate.

TABLE 5 In vitro proteasomal cleavage of a 29 amino acid long HA-1Rpeptide % fragment digested in 15 min 30 min 45 min G L E K L K E C V L  R  D  D L L E A R R P R A H E C L G E A 26.2 28.0 23.8 G L E K L KE C V L R D D L L E A R R P R A H E C L G 14.0 16.0 13.5 G L E K L K E CV L R D D L L E A R R P R A H E C L G E 11.1 14.3 12.7 G L E K L K E C VL R D D L L  7.9 9.6 7.9 G L E K L K E C V L R D D L L E A R R P R A 6.6 8.6 8.3     E K L K E C V L R D D L L  6.2 7.5 7.3               CV L R D D L L E A R R  5.3 7.0 5.7 G L E K L K E C V L R D D L L E A R RP R A H E C L  4.9 7.1 6.4 G L E K L K E C V L R D D L L E A R R P R AH E C  4.2 6.1 5.6 G L E K L K E C V L R D  3.9 4.2 3.9 G L E K L K E CV L R D D L L E A  3.6 4.4 4.4 G L E K L K E C V L R D D L L E A R R 3.4 4.2 3.6 G L E K L K E C V L R D D L L E A R R P R  2.6 4.2 4.0 G LE K L K E C V L R D D L L E A R R P R A H The peptide sequences thatbind to HLA-B60 are underlined. The proteolytic fragments cleaved at theCOOH termini of the HLA-B60 binding peptides are indicated in bold. Theamounts of the generated fragments after cleavage with 20s immunoproteasomes for 15, 30 and 45 minutes are expressed as the percentage ofall fragments found in the digested substrate.

TABLE 6 CTL DNA analysis Number analysis HA-1 of clones HA-1 Cellphenotype KIAA0223 sequence sequenced phenotype DH HA-1−/−GAGTGTGTGTTGCGTGACGACCTCCTTGAGGCCCGCCG (6/6 clones) HA-1^(R)/HA-1^(R)E  C  V  L  R  D  D  L  L  E  A  R  R vR HA-1+/+GAGTGTGTGCTGCATGACGACCTCCTTGAGGCCCGCCG (6/6 clones) HA-1^(H)/HA-1^(H)E  C  V  L  H  D  D  L  L  E  A  R  R KG-1 HA-1+GAGTGTGTGTTGCGTGACGACCTCCTTGAGGCCCGCCG (1/8 clones) HA-1^(R)/HA-1^(H)E  C  V  L  R  D  D  L  L  E  A  R  RGAGTGTGTGCTGCATGACGACCTCCTTGAGGCCCGCCG (7/8 clones)E  C  V  L  R  D  D  L  L  E  A  R  R

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1. A peptide characterized in being immunogenic and obtainable from aminor Histocompatibility antigen HA-1, said peptide furthercharacterized by comprising a sequence selected from the group ofsequences consisting of VLXDDLLEA (SEQ ID NO:1), KECVLXDDL (SEQ IDNO:3), combinations thereof, and a derivative of any thereof havingsimilar functional or immunological properties, wherein X represents ahistidine or an arginine residue.
 2. The peptide of claim 1, wherein thesequence is VLHDDLLEA (SEQ ID NO:2).
 3. The peptide of claim 1, whereinthe sequence is KECVLHDDL (SEQ ID NO:4).
 4. A preparation comprising thepeptide of claim
 1. 5. A preparation comprising the peptide of claim 2.6. A preparation comprising the peptide of claim
 3. 7. A method ofinducing tolerance in a subject to transplants to prevent rejectionand/or Graft versus Host disease or a method treating (auto)immunedisease in a subject, said method comprising: administering thepreparation of claim 4 to the subject.
 8. A method for elimination of agroup of hematopoietic cells, said method comprising: presenting thepeptide of claim 1 in the context of HLA class-I, wherein saidelimination is induced directly or indirectly by specific recognition ofthe peptide in the context of HLA class-I.
 9. An analog of the peptideof claim 1, wherein said analog is an antagonist for the activity of aT-cell recognizing the peptide.
 10. A process for producing antibodies,T-cell receptors, anti-idiotypic B-cells, T-cells, or mixtures of anythereof, said process comprising: immunizing a mammal with the peptideof claim 1; and harvesting antibodies, T-cell receptors, anti-idiotypicB-cells, T-cells, or mixtures of any thereof from the mammal. 11.Antibodies, T-cell receptors, B-cells, T-cells, and or mixtures of anythereof obtainable by the process of claim
 10. 12. A process forgenerating a cytotoxic T-cell against a minor antigen, said methodcomprising: contacting a cell selected from the group of a hematopoieticcell and a dendritic cell with the peptide of claim 1, thus generating acytotoxic T-cell against the minor antigen.
 13. The process of claim 12,wherein the cell is contacted with the peptide in the context ofHLA-B60.
 14. The process of claim 12, wherein the cell is a dendriticcell.
 15. The process of claim 12, wherein the cell is a hematopoieticcell negative for said minor antigen.
 16. The process of claim 12,wherein said minor antigen is HA-1.
 17. The process of claim 12, whereinthe contacting is carried out ex vivo.
 18. The process of claim 12,wherein said cytotoxic T-cell includes a suicide gene.
 19. The processof claim 12, wherein said cytotoxic T-cell is immortalized.
 20. Acytotoxic T-cell obtainable by the process claim
 12. 21-48. (canceled)